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US5477001A - Recombinant DNA coding for a novel protein having β-1,3-glucanase activity, bacteria containing this DNA, transformed plant cells and plants - Google Patents

Recombinant DNA coding for a novel protein having β-1,3-glucanase activity, bacteria containing this DNA, transformed plant cells and plants Download PDF

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US5477001A
US5477001A US07/966,187 US96618793A US5477001A US 5477001 A US5477001 A US 5477001A US 96618793 A US96618793 A US 96618793A US 5477001 A US5477001 A US 5477001A
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Catherine Sass
Jean-Jacques Leguay
Rene Grison
Alain Toppan
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Societe National Elf Aquitaine
Rustica Prograin Genetique
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Elf Sanofi SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01039—Glucan endo-1,3-beta-D-glucosidase (3.2.1.39)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01058—Glucan 1,3-beta-glucosidase (3.2.1.58)

Definitions

  • the invention relates to a recombinant DNA coding for a novel protein having ⁇ -1,3-glucanase activity, or for a precursor of this protein, to a bacterium containing this recombinant DNA, to a plant cell, plant or plant part, especially a plant seed, transformed by this recombinant DNA, as well as to this novel protein and to a method for preparing it.
  • Crop plants are known to be subjected to attack by-parasites such as phytopathogenic fungi, which are responsible for substantial harvest losses.
  • the .main means at present for controlling these fungi lies in the use of chemical substances having fungicidal activity. It is now known that plants react naturally to such attack by various defence mechanisms, which are unfortunately, in general, triggered too late and at too low an intensity to be sufficiently effective.
  • One of these mechanisms comprises the induction of an enzyme known as ⁇ -1,3-glucanase E.C.3.2.1.39 (Kombrink et al., 1988, Pr. Ntl. Acad. Sci. USA, 85,982-986 and Pegg et al., 1981, Physiol. Plant. Pathol. 19,371-382).
  • This induction can be triggered artificially through the effect of elicitors, that is to say compounds of biological origin capable of inducing in a healthy plant the defence reactions which it deploys naturally during an infection by pathogenic agents, or during a hormonal imbalance caused by auxin, cytokinins or ethylene (Abeles et al., 1971, Plant Physiol. 47,129-134 and de Loose et al., 1988, Gene, 70,13-23).
  • ⁇ -1,3-glucans are linear polysaccharide polymers consisting of glucose units linked via ⁇ -(1 ⁇ 3) linkages, sometimes possessing ⁇ -(1 ⁇ 4) or ⁇ -(1 ⁇ 6) type branching (Farka 1982, in "Fungal protoplasts", Peberdy and Ferencry, published by Dekker Inc.). These polysaccharides constitute a typical component of the skeleton of the wall of most fungi, and in particular phytopathogenic fungi.
  • ⁇ -1,3-glucanases are capable of degrading them by fragmentation of the ⁇ -1,3-glucan chains. Most known plant ⁇ -1,3-glucanases are of the endo type.
  • DNA sequences coding for several plant ⁇ -1,3-glucanases in particular a Nicotiana plumbaginifolia ⁇ -1,3-glucanase (De Loose et al., 1988, Gene, 70,13-23) and a soybean ⁇ -1,3-glucanase (Takeuchi et al., 1990, Plant Physiol., 93,673-682), have been isolated, cloned and determined.
  • Patent Application EP-A-0,353,191 describes the isolation and cloning of different fragments of complementary DNA, the assembling of which enables the complementary DNA sequence coding for a tobacco ⁇ -1,3-glucanase to be deduced, as well as of the genomic DNA sequence coding for this enzyme.
  • the document EP-0,392,225 discloses, in particular, the construction of a chimeric gene coding for this tobacco ⁇ -1,3-glucanase, the transformation of tobacco with the latter and the verification, by Western blot and visualisation using polyclonal antibodies, of the over-expression of this endogenous protein in the transformed plants.
  • This patent application does not show that the recombinant tobacco ⁇ -1,3-glucanase is biologically active, nor a fortiori that it confers a resistance of the said transformed plants to pathogenic agents.
  • ⁇ -1,3-glucanases such as, for example, Bacillus subtilis ⁇ -1,3-glucanase are useful enzymes in the conversion of biomass, in particular in some sectors of the papermaking industry and in the agri-foodstuffs industry, especially brewing (Meaden, 1986, Brewers Guardian, 115, 7 and MacQueen, October 1987, New Scientist, 66).
  • the present invention relates to a novel recombinant DNA, characterised in that it codes for a protein having ⁇ -1,3-glucanase activity or for a precursor thereof, this protein having ⁇ -1,3-glucanase activity comprising the following amino acid sequence (a 1 ) (SEQ ID NO:1):
  • a high degree of homology means here a homology (ratio of identical amino acids to the total number of amino acids) of at least 80%, and preferably at least 90%, of the amino acid sequences when they are aligned on the basis of maximal homology according to the optimal sequence alignment method of Needleman and Wunsch, 1970, J. Mol. Biol., 48, 443-453. This method is used, in particular, in the University of Wisconsin's UWGCG software : Devereux et al., 1984, Nucl. Ac. Res., 12, 8711-8721--GAP option.
  • the already known peptide sequence closest to that of the sequence (a 1 ) (SEQ ID NO.1): of 307 amino acids is that of the protein of 369 amino acids deduced from the complementary DNA of a Nicotiana plumbaginifolia ⁇ -1,3-glucanase SEQ ID NO.12) (see Swissprot data bank ref. Gus$ Nipl. and De Loose et al., 1988, Gene, 70, 13-23).
  • This novel recombinant DNA may be used for the expression of this protein having ⁇ -1,3-glucanase activity, either to confer an enhanced resistance to pathogenic agents on a plant or plant part expressing the protein, or to produce this protein using eukaryotic cells, in particular ascomycetes such as yeast or filamentous fungi, for example Cryphonectria parasitica, or plant cells, or prokaryotic microorganisms such as, for example, Escherichia coli.
  • eukaryotic cells in particular ascomycetes such as yeast or filamentous fungi, for example Cryphonectria parasitica, or plant cells, or prokaryotic microorganisms such as, for example, Escherichia coli.
  • This recombinant DNA can contain, immediately downstream of the nucleotide sequence coding for the amino acid sequence (a 1 ) (SEQ ID NO1), the nucleotide sequence coding for the following amino acid sequence (a 4 ) (SEQ ID NO:4):
  • this recombinant DNA comprises immediately upstream of the nucleotide sequence coding for the amino acid sequence (a 1 ) (SEQ ID NO:1), a codon for Gln.
  • An especially high valued DNA such as defined above is that which codes for a protein having ⁇ -1,3-glucanase activity or a precursor thereof, and which comprises the following sequence (a 5 ) (SEQ ID NO:3):
  • This recombinant DNA preferably contains, upstream of the sequence coding for the sequence (a 1 ) (SEQ ID NO:1) optionally preceded by a codon for Gln, a signal sequence, chosen in accordance with the host cell, which has the function of enabling the protein to be exported from the cytoplasm.
  • this signal sequence can be either a sequence derived from a sequence coding for a natural precursor of a protein exported by a prokaryotic microorganism, for example the OmpA signal peptide (Ghrayeb et al., 1984, EMBO Journal, 3, 2437-2442) or an alkaline phosphatase precursor (J. Bact.
  • sequence coding for a eukaryotic precursor for example the signal peptide of one of the natural precursors of human growth hormone
  • sequence coding for a synthetic signal peptide for example that described in French Patent Application No. 2,636,643, of sequence (SEQ ID NO:4):
  • this signal sequence is preferably a sequence derived from a sequence coding for a natural precursor of a protein secreted by these cells, for example, for yeast, the invertase precursor (Patent Application EP-0,123,298) or the precursor of the prepro sequence of the alpha pheromone (Patent Application DK 2484/84), or for Cryphonectria parasitica, the precursor of the prepro sequence of endothiapepsin (described in French patent application no. 2 666 590), of sequence (SEQ ID NO: 5):
  • amino acid sequences (a 1 ), (a 2 ) and (a 4 ), (SEQ ID NOS:1, 7 and 2, respectively) can, for example, be encoded by the nucleotide sequences (Na 1 ) (SEQ ID NO:8), (Na 2 ) and (Na 4 ) below:
  • the invention also relates to a unit for expression of the recombinant DNA defined above, advantageously carried out by a vector, termed expression vector.
  • the recombinant DNA For an expression in procaryotic microorganisms, especially in Escherichia coli, the recombinant DNA must be inserted in an expression unit containing, in particular, an effective promoter, followed by a ribosome binding site upstream of the gene to be expressed, as well as an effective transcription stop sequence downstream of the gene to be expressed.
  • This unit must also contain a selection marker or be introduced into the host cell at the same time as a unit for expression of a selection marker (for example using an expression vector which carries both of these units). All these sequences must be chosen in accordance with the host cell.
  • the expression unit according to the invention comprises the recombinant DNA defined above with the means needed for its expression.
  • the expression unit carries a selection marker or is introduced into the host cell at the same time as a selection marker.
  • this selection marker is an auxotrophic marker (which complements a mutation of the recipient cells) which permits the selection of cells which have integrated the recombinant DNA in a high copy number, either into their genome or into a multicopy vector.
  • the expression unit according to the invention carries the recombinant DNA defined above with the means needed for its expression, and optionally a selection marker and/or telomeric sequences. It is, in effect, possible to select transformants which have integrated a DNA of interest using a selection marker located either on the same unit as the DNA of interest or on another unit, these two units then being introduced by cotransformation.
  • the recombinant DNA of the invention may be either integrated in the genome of the filamentous fungi, or preserved in extrachromosomal form by means of sequences enabling this DNA to be replicated and split.
  • the promoter is preferably a strong constitutive promoter, for example the 35S promoter of the cauliflower mosaic virus, or a promoter controlling the tissue- or organ-specific expression, such as the promoter of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase, which is expressed preferentially in the leaves and most especially the mesophyll tissues (Kuhlemeier et al., 1987, Ann Rev Plant Physiol 38: 221-257).
  • a specific promoter controlling for example, an expression in the seeds or during a precise stage of development of the plant, or a promoter which is inducible following a thermal shock, a wound or interaction between the plant and parasites (Kuhlemeier et al., 1987, reference cited above), if an expression of recombinant DNA is desired in these situations.
  • the terminator sequence containing polyadenylation sites which can be isolated from plant genes or from genes which are expressed in plants, such as, for example, the nopaline synthase terminator Agrobacterium tumefaciens, is used.
  • the invention also relates to a bacterium, for example of the species E. coli, which contains the recombinant DNA defined above with the means needed for its replication and its expression.
  • This bacterium may be used in the preparation of a protein having ⁇ -1,3-glucanase activity.
  • the invention also relates to a bacterium, for example of the species E. coli, which contains the recombinant DNA defined above with the means enabling it to be replicated, which bacterium may hence be used for the cloning of this recombinant DNA, and also to a bacterium capable of infecting a plant with transfer of genetic material, for example of one of the species Agrobacterium rhizogenes and Agrobacterium tumefaciens, which contains this DNA in a context enabling it to be replicated and may hence be used for transforming plant cells.
  • a bacterium for example of the species E. coli, which contains the recombinant DNA defined above with the means enabling it to be replicated, which bacterium may hence be used for the cloning of this recombinant DNA
  • a bacterium capable of infecting a plant with transfer of genetic material for example of one of the species Agrobacterium rhizogenes and Agrobacterium tum
  • the transformation of plant cells with the above recombinant DNA may also be performed by another biological method such as the pollen tube method (Zhong-xun Luo et al., Plant Molec. Biol. Rep., 1988, 6, 165-176) and the direct transformation of germinating seeds (Toepfer R. et al., 1989, The Plant Cell., 1, 133-139), or by a physical method such as the use of polyethylene glycol, electroporation (Chistou P. et al., 1987, Proc. Ntl. Acad. Sci. USA, 84, 3662-3699) and bombardment using microprojectiles (Klein T. M. et al., 1988, Proc. Ntl. Acad. Sci. USA, 85, 8502-8505).
  • another biological method such as the pollen tube method (Zhong-xun Luo et al., Plant Molec. Biol. Rep., 1988, 6, 165-176) and the direct transformation of germinating seeds
  • the invention also relates to a plant cell, characterised in that it is transformed by the recombinant DNA defined above with the means needed for its expression.
  • This plant cell can originate from a major crop species such as, for example, maize, soybean, beet, wheat, barley, poppy, rape, sunflower, alfafa and sorghum, a floral species such as rose, carnation and gerbera or a culinary species such as carrot, tomato, lettuce, chicory, pepper, melon and cabbage.
  • a major crop species such as, for example, maize, soybean, beet, wheat, barley, poppy, rape, sunflower, alfafa and sorghum, a floral species such as rose, carnation and gerbera or a culinary species such as carrot, tomato, lettuce, chicory, pepper, melon and cabbage.
  • Especially highly valued species are Brassica napus rape, Helianthus annuus sunflower and Nicotiana tabacum tobacco.
  • the step of transformation which involves one or a few cells is followed by a step of multiplication of these transformed cells so as to obtain calluses, which can give rise to transformed plants by processes of organogenesis or embryogenesis.
  • the invention hence also relates to a plant or plant part, characterised in that it contains the recombinant DNA defined above with the means needed for its expression.
  • An especially highly valued plant part is the part capable of forming a complete new plant, in particular after sowing, burying in the ground or transplanting, or of producing seeds.
  • Such a plant part is, for example, a grain, a mature fertilised ovule, a seed, a cutting, a runner, and the like.
  • These plants can be any one of the above species, and more especially of the species Nicotiana tabacum, Helianthus annuus and Brassica napus.
  • the invention also relates to a method for obtaining plants resistant to parasites, such as phytopathogenic fungi, which comprises a step of transformation of plant cells with this recombinant DNA, followed by a step of multiplication of the transformed cells and a step of regeneration of the plants.
  • parasites such as phytopathogenic fungi
  • the step of transformation of the plant cells is performed in vitro using an agrobacterium (that is to say a bacterium of the genus Agrobacterium) which has integrated recombinant DNA of interest.
  • an agrobacterium that is to say a bacterium of the genus Agrobacterium
  • the invention also relates to plants resistant to pathogenic agents, which are capable of being obtained using the method defined above.
  • the invention also relates to the use of a plant containing the recombinant DNA defined above with the means needed for its expression, as a parent in a selection programme for creating new plant varieties.
  • the invention also relates to a novel protein having ⁇ -1,3-glucanase activity, which comprises the sequence (a 1 ) (SEQ ID NO:1) and optionally, immediately downstream of the sequence (a 1 ), the sequence (a 4 ) (SEQ ID NO:2) truncated in its carboxy-terminal portion by 0 to 27 amino acids, and to a novel protein comprising the sequence (a 5 ) (SEQ ID NO:3).
  • This protein preferably possesses an apparent molecular mass of 36 ⁇ 3, 37 ⁇ 3 or 39 ⁇ 3 kDa.
  • This protein is of interest as an enzyme for conversion of biomass which contains ⁇ -1,3-glucans, in particular in some sectors of the papermaking industry and in the agri-foodstuffs industry, especially brewing.
  • the invention also relates to a method for preparing this protein, which comprises the culturing of bacteria, plant cells, plant calluses, plants or plant parts containing the recombinant DNA defined above, lysis thereof and isolation and purification of this protein.
  • FIG. 1 shows a restriction map of the HindIII-EcoRI fragment contained in plasmid pBR 1310 and containing the complementary DNA coding for soybean ⁇ -1,3-glucanase.
  • FIGS. 2A-2D show the nucleotide sequence (SEQ ID NO:11) of the HindIII-HindIII insert, part of the complementary DNA of soybean ⁇ -1,3-glucanase, the restriction sites used for cloning in M13mp19 and for the subsequent constructions being indicated by arrows, as well as the peptide sequence (SEQ ID NO:12) deduced from this complementary DNA.
  • FIGS. 3A-3D show the nucleotide sequence (SEQ ID NO:13) of the HindIII-HindIII fragment of the complementary DNA of soybean ⁇ -1,3-glucanase after mutagenesis (at the end of section 6c), the restriction sites used for cloning in M13mp19 and for the subsequent constructions being indicated by arrows, as well as the peptide sequence of the protein translated.
  • FIGS. 4A-4C show the nucleotide sequence (SEQ ID NO:15) of the NdeI-HindIII fragment of the coding portion of plasmid pBR59, an expression plasmid in E. coli, flanked by NdeI and HindIII restriction sites, and the peptide sequence (SEQ ID NO:16): of the protein translated.
  • FIGS. 5A-5E show the nucleotide sequence (SEQ ID NO:17) of the complete recombinant gene and the peptide sequence of (SEQ ID NO:18) of the protein translated.
  • FIGS. 6A-6B show the peptide sequence (SEQ ID NO:25) of the common portion of the proteins translated in E, coli and in tobacco.
  • FIGS. 7A and 7B show an alignment, on the basis of the method of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443-453, of the peptide sequence (SEQ ID NO:12) deduced from the complementary DNA of soybean ⁇ -1,3-glucanase (see FIGS. 2A-2D) and the closest known peptide sequence (SEQ ID NO:26), that deduced from the complementary DNA of Nicotiana plumbaginifolia ⁇ -1,3-glucanase (Swissprot data bank ref. Gub$-Nipl).
  • Section1 Preparation of polyclonal antibodies against tobacco ⁇ -1,3-glucanase
  • a tobacco ⁇ -1,3-glucanase was purified to homogeneity from tobacco calluses as described below.
  • Tobacco calluses were cultured in vitro on a Murashige and Skoog medium (Murashige T. and Skoog F., 1962, Physiol. Plant., 15, 473-497).
  • Cell extracts are obtained by grinding the plant material in a 50 mM Tris-HCl buffer solution, pH 8.4, containing 15 mM ⁇ -mercaptoethanol and 5% of polyvinylpyrrolidone.
  • the protein is purified from this extract by ammonium sulphate precipitation, liquid chromatography on a cation exchange column based on synthetic polymer and exclusion chromatography (molecular sieving) on a crosslinked agarose, according to the protocol described below.
  • the protein extract is treated with ammonium sulphate (43% saturation).
  • the proteins which have precipitated are collected by centrifugation (15,000 g for 30 min), solubilised in a buffer solution (100 mM ammonium acetate, pH 5.2) and dialysed overnight at 4° C. against a 100 mM ammonium acetate buffer solution, pH 5.2.
  • the acetate concentration of the buffer solution of the protein extract is brought to 10 mM by passage through ready-to-use minicolumns (Pharmacia PD-10).
  • the protein extract is then purified by ion exchange chromatography using a column based on synthetic polymer (Pharmacia Mono-S column) according to the Pharmacia FPLC technique.
  • the extract is applied to a Mono-S column equilibrated with a 10 mM ammonium acetate buffer, pH 5.2.
  • the proteins retained on the column are eluted with a linear gradient from 10 to 500 mM ammonium acetate.
  • the ⁇ -1,3-glucanase is identified by its molecular weight (polyacrylamide gel electrophoresis in the presence Of SDS--visualisation with silver), and its activity is measured by a colorimetric method (see section 8 below) using laminarin ( ⁇ -1,3-glucan extracted from Laminaria digitataz--Sigma--ref. L9634) as a substrate.
  • Rabbits were then injected with 25 ⁇ g of tobacco ⁇ -1,3-glucanase in 500 ⁇ l of Freund's complete adjuvant. Three booster injections of 25 ⁇ g in Freund's incomplete adjuvant (500 ⁇ l) were carried out at 3-week intervals. The immune serum was drawn 3 weeks after the final injection.
  • This immune serum recognises tobacco ⁇ -1,3-glucanase; it also recognises soybean ⁇ -1,3-glucanase. It was, in effect, verified that it enables the latter protein to be visualised by the Western blot technique (described in section 13 below) from an extract of total soybean (Glycine max cv Mandarin) proteins.
  • Section 2 Construction of a phage library of complementary DNA from messenger RNAs of soybean cell cultures.
  • RNA of 5-day-old soybean cells (Glycine max cv Mandarin), cultured in vitro in the absence of auxin according to the method described by Leguay et al., 1987, Develop. Genetics 8: 351-364, was extracted according to the method described by Jouanneau et al., 1984 Plant Physiol 74; 663-668, summarised below.
  • the cells washed beforehand in saline solution, are ground in liquid nitrogen; the homogenate is then extracted with a mixture of redistilled phenol and chloroform. After an ethanol precipitation step, the total RNA is dissolved in a solution buffered to pH 7.6.
  • the poly(A) + fraction of the messenger RNA (mRNA) was isolated after 2 cycles of affinity chromatography on an oligo(dT)-cellulose column as described in Sambrook et al., ("Molecular cloning: A Laboratory manual", second edition, Cold Spring Harbor Laboratory 1989). Quantification of the messenger RNAs is carried out by spectrophotometry according to a protocol well known to a person skilled in the art.
  • cDNAs Complementary DNAs
  • the synthesised double-stranded cDNA is then methylated using EcoRI methylase under the conditions described in the reference work by Sambrook et al. (op. cit.).
  • This enzyme enables the possible EcoRI sites of the cDNA to be protected against cleavage by the endonuclease EcoRI, this protection disappearing for the replicates of the cDNA (which are not methylated).
  • Synthetic EcoRI linkers double-stranded DNA fragments containing the EcoRI site
  • Synthetic EcoRI linkers double-stranded DNA fragments containing the EcoRI site
  • the cDNA is ligated using T4 DNA ligase in phage lambda gt11, a cloning and expression vector described by Huynh et al., "DNA cloning: a practical approach" IRL Press, D. M. Glover 1985, p. 49, according to the Promega protocol (ref.
  • T3011) "lambda gt11 system", the phage DNA being opened beforehand with the restriction endonuclease EcoRI and dephosphorylated. Aliquot portions of the ligation medium are then packaged in phage particles using the kit marketed by Amersham ("In vitro packaging system for lambda DNA", ref. N334).
  • the number of recombinants is then estimated by counting the lytic plaques obtained on a bacterial lawn of E. coli strain Y1090 (Sambrook et al., ref. above, op. cit.). Approximately 10 6 clones were obtained.
  • the production of a library in the vector lambda gt11 makes it possible to carry out the expression of the cloned cDNAs, that is to say to synthesise the proteins encoded by the messenger RNAs which were used for constructing this library.
  • This synthesis takes place after induction with IPTG (isopropyl ⁇ -D-thiogalactoside); the synthesised proteins can then be recognised by the antibodies obtained against tobacco ⁇ -1,3-glucanase (see section 1).
  • IPTG isopropyl ⁇ -D-thiogalactoside
  • the clones producing these proteins can then be identified and isolated according to a protocol known to a person skilled in the art and described, for example, in Sambrook et al. (op. cit.).
  • TNT solution a solution composed of 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% of Tween 20 detergent
  • blocking buffer a buffer composed of gelatin in 1% solution in TNT solution.
  • the filters are then treated for 3 h in a solution containing the anti- ⁇ -1,3-glucanase polyclonal antibodies obtained above (see Section 1), diluted in the blocking buffer. They were then immersed for 10 minutes successively in each of the following solutions:
  • the antigen-antibody complex formed is then visualised using secondary antibodies conjugated by coupling with a peroxidase ("Immuno conjugate GAR/IgG (H+L)/Po" of Nordic Immunology, Tilburg, Holland).
  • the chemical visualisation reaction employs 4-chloro-1-naphthol as a chromogenic substrate (giving a blue precipitate).
  • the positive lyric plaques that is to say those corresponding to clones which synthesise ⁇ -1,3-glucanase, are then identified on the Petri dish, and the bacteriophages are removed and purified by means of a secondary immunological screening, performed in a strictly identical manner to the primary screening. One of these clones was selected for continuation of the study.
  • Section 4 Partial characterisation of the phage DNA of the soybean ⁇ -1,3-glucanase clone.
  • the selected phage clone is amplified and its DNA is extracted according to the protocol described in the Amersham kit "cDNA cloning system GT11" (ref. RPN 1280), summarised below.
  • the phages are removed using a coring tool and transferred to a culture of E. coli strain Y1090; after 15 min, 5 ml of Luria medium (Gibco) containing 5 mM of CaCl 12 and 50 ⁇ g/ml of ampicillin are added. After incubation for 4 h at 43° C. with agitation, the culture is centrifuged at 3,000 rpm for 10 min.
  • the DNA of the phage particles present in the supernatant is then extracted and purified by treatment with polyethylene glycol and sodium chloride NaCl, centrifugation, treatment with proteinase K and precipitation.
  • DNA of the ⁇ -1,3-glucanase clones was hydrolysed with the restriction enzyme EcoRI.
  • EcoRI restriction enzyme
  • Section 5 Construction of plasmid pBR1310, isolation of the complementary DNA insert coding for soybean ⁇ -1,3-glucanase and determination of its sequence.
  • Plasmid pGEM-3Z (Promega, Madison, Wis, U.S.A.) was opened using the restriction endonucleases EcoRI and SmaI, then purified and isolated by electrophoresis on low-melting-temperature agarose gel.
  • This plasmid comprises a "polylinker” (cloning multisite) successively containing EcoRI, SmaI and HindIII restriction sites.
  • plasmid pBR1310 ligation of the SmaI and PvuII blunt ends with disappearance of these sites
  • the vector obtained was then extracted and purified by the alkaline lysis method (Birnboim and Doly, in Sambrook et al., op. cit.).
  • the HindIII-HindIII fragment (of approximately 1490 bp) was prepared by the digestion of the HindIII-EcoRI fragment with the endonuclease HindIII, purified by electrophoresis on low-melting-temperature agarose gel and isolated. This fragment was cloned into the DNA of the replicative form of the single-stranded phage M13mp19 (Pharmacia) opened at the HindIII restriction site.
  • the vector M13 containing the 1490-bp HindIII-HindIII insert is referred to as M13BR137. The sequence of this insert is determined according to the dideoxyribonucleotide method (Sanger et al., PNAS-USA, 14, 5463-5467, 1977).
  • FIGS. 2A-2D A better understanding of the description below will be gained with the aid of FIGS. 2A-2D.
  • This sequence contains a single open reading frame (not interrupted by a stop codon) compatible with the apparent molecular weight observed by agarose gel electrophoresis: the sequence beginning with an ATG codon at position 114 and terminating with the TAA stop codon at position 1224-1226 coding for a protein of 370 amino acids.
  • a signal peptide is expected by a person skilled in the art, since ⁇ -1,3-glucanases are proteins which can be naturally either accumulated in the vacuoles of plant cells or secreted, thereby demanding the presence of a signal peptide.
  • the already-known peptide sequence closest to that of the protein of 370 amino acids deduced from the complementary DNA of soybean ⁇ -1,3-glucanase is that of the protein of 369 amino acids deduced from the complementary DNA of a Nicotiana plumbaginifolia ⁇ -1,3-glucanase (SEQ ID NO:22) (see Swissprot data bank ref. Gub$Nipl. and De Loose et al., 1988, Gene, 70, 13-23).
  • ⁇ -1,3-glucanases are known at present, the amino-terminal sequences of which have been determined experimentally. They are the ⁇ -1,3-glucanases of Nicotiana tabacum (H. Shinshi et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5541-5545), of Nicotiana plumbaginifolia (M. De Loose et al., 1988, Gene, 70, 13-23), of Phaseolus vulgaris bean (B. V. Edington et al., 1991, Plant Molecular Biology, 16, 81-94) and of Hordeum vulgare barley (P. B. Hoj et al., 1988, Febs Lett., 230, 67-71).
  • Section 6 Construction of a vector for expression
  • sequence GAG CTC of the SacI restriction site located at position 575 of the vector M13BR137 is replaced, by directed mutagenesis as described below, by the sequence GAG CAC.
  • the codon GCT corresponding to an alanine residue in the initial cDNA sequence, is hence replaced by the codon GCA, also corresponding to an alanine residue (silent mutation).
  • the single-stranded form of the vector M13BR137 is isolated according to the protocol described by Sambrook et al., op. cit., from a culture of the E. coli bacterial strain DH5 ⁇ F' (Gibco-BRL) transformed beforehand with this vector according to the protocol recommended by the manufacturer.
  • mutagenesis is performed according to the protocol of the "Oligonucleotide-directed in vitro mutagenesis system" kit (Amersham--ref. RPN 1523--version 2), using an oligonucleotide synthesised chemically on the Millipore Biosearch 8700 DNA synthesiser, the sequence of which is shown below (SEQ ID NO:19):
  • the directed mutagenesis technique described in detail in the booklet accompanying this kit, and summarised below, consists in hybridising the above oligonucleotide with the single-stranded form of the vector M13BR137, and then in reacting Klenow polymerase and T4 DNA ligase in the presence of the thionucleotide dCTP- ⁇ -S ( ⁇ -thiodeoxycytidine triphosphate) so as to obtain a circular double-stranded form of the recombinant phage, one of the strands of which carries the desired mutation and is protected from cleavage by the endonuclease NciI and degradation by exonuclease III. On the other strand (which has served as a template) a break is then introduced using the endonuclease NciI, and this strand is thereafter removed by the subsequent action of exonuclease III.
  • vector M13BR138 The resulting vector, referred to as vector M13BR138, was cloned into E. coli strain DH5 ⁇ GF' and sequenced; it possesses, as expected, a sequence mutated by replacement of a codon GCT by a codon GCA, which has eliminated the SacI restriction site from the coding portion.
  • the single-stranded form of the vector M13BR138 is isolated as described above, and a directed mutagenesis is then carried out under the same conditions as before using the chemically synthesised oligonucleotide the sequence of which is noted below (SEQ ID NO:20)
  • vector M13BR139 The vector derived from this mutagenesis, referred to as vector M13BR139, is cloned into the E. coli bacterial strain DH5 ⁇ F'; it was sequenced, which confirmed the introduction of NdeI and BamHI restriction sites upstream of the ATG translation initiation codon.
  • the single-stranded form of the vector M13BR139 is isolated, and a directed mutagenesis is then carried out on the 3'portion of the insert using the oligonucleotide of the following sequence and under the conditions already described SEQ ID NO:21:
  • vector M13BR140 The vector resulting from this mutagenesis, referred to as vector M13BR140, is cloned into the E. coli bacterial strain DH5 ⁇ F'. Its sequence was determined, which confirmed the creation of HindIII and SacI restriction sites downstream of the TAA stop codon. This sequence is shown in FIGS. 3A-3D and in SEQ ID NO:13.
  • the replicative form of the vector M13BR140 is isolated and purified according to the protocol of Birnboim and Doly described in Sambrook et al., op. cit.
  • the NdeI-HindIII insert containing the ⁇ -1,3-glucanase coding portion was isolated by digestion with the restriction enzymes NdeI and HindIII, and purified by agarose gel electrophoresis and electroelution (Sambrook et al., op. cit.). This insert is then ligated using T4 DNA ligase in the large NdeI-HindIII fragment of plasmid p373,2, the construction and constitutive elements of which are described in Patent Application EP-A-0,360,641 (FIGS.
  • 3A-3D of this document shows the restriction map of this plasmid), opened beforehand by digestion with the restriction enzymes NdeI and HindIII.
  • This large fragment contains, in particular, successively from the NdeI end to the HindIII end, a promoter analogous to the "tac" promoter (Sambrook et al., op. cit.) which is inducible with isopropyl ⁇ -D-galactoside, a gene coding for ⁇ -lactamase (conferring resistance to ampicillin) in a context permitting its expression, an origin of replication in E. coli and a transcription terminator derived from the phage fd.
  • the vector obtained, referred to as plasmid pBR141 is cloned into E. coli strain RR1. The structure of the plasmid is verified by producing a restriction map.
  • the plasmid DNA of the clone containing plasmid pBR141 is extracted according to the protocol of Birnboim and Doly (Sambrook et al., op. cit.).
  • the first amino acid of this protein namely the glutamine residue (capable of cyclising to pyroglutamate, which makes it impossible to determine the amino-terminal sequence), being replaced by a translation initiation methionine residue.
  • plasmid pBR59 plasmid pBR59
  • plasmid pBR59 plasmid pBR59
  • plasmid pBR59 plasmid pBR59
  • tac tac promoter
  • IPTG isopropyl ⁇ -D-thiogalactoside
  • Section 7 Expression of soybean ⁇ -1,3-glucanase in E. coli.
  • the E. coli strain RR1 containing the expression vector pBR59 and an E. coli strain RR1 containing plasmid p373,2 are cultured in Luria medium (Gibco) containing 100 mg/1 of the antibiotic ampicillin overnight at 37° C. After a dilution of the culture to 1/100 in the same medium, the bacteria are returned to culture for 1 hour at 37° C. and IPTG is then added at a final concentration of 1 mM. Culturing is then continued for 3 hours and the bacteria are harvested by centrifugation.
  • Luria medium Gibco
  • loading buffer of the following composition:
  • Section 8 Measurement of the enzymatic activity of the recombinant soybean ⁇ -1,3-glucanase expressed in E. coli, isolation and purification of three forms of this protein and determination of their amino-terminal sequences.
  • ⁇ -1,3-glucanase activity is measured by a colorimetric method enabling the amount of monomers or oligomers liberated by the enzyme from a substrate (laminarin) to be estimated by determining the reducing power of the sugars thus liberated.
  • This method described by G. Ashwell, 1957, in “Methods in Enzymology III", 73-105, S. P. Colowick and N. U. Kaplan Eds., is summarised below.
  • ⁇ -1,3-glucan extracted from Laminaria digitata--Sigma--ref. L9634 50 ⁇ l of a solution containing 50 mg/ml of laminarin ( ⁇ -1,3-glucan extracted from Laminaria digitata--Sigma--ref. L9634) are added to a solution containing a concentration of ⁇ -1,3-glucanase chosen so as to fall within a linear response range.
  • the volume of the reaction mixture is adjusted to 500 ⁇ l using a 0.2M sodium acetate buffer solution, pH 5.0.
  • Somogyi's reagent mixture of 25 ml of an aqueous solution comprising 2.5% of Na 2 CO 3 , 2.5% of KNaC 4 H 4 O 6 .4H 2 O, 2% of NaHCO 3 , 20% of Na 2 SO 4 and 1 ml of aqueous solution containing 15% of CuSO 4 .5H 2 O
  • Somogyi's reagent mixture of 25 ml of an aqueous solution comprising 2.5% of Na 2 CO 3 , 2.5% of KNaC 4 H 4 O 6 .4H 2 O, 2% of NaHCO 3 , 20% of Na 2 SO 4 and 1 ml of aqueous solution containing 15% of CuSO 4 .5H 2 O
  • the tubes are then cooled in ice before adding 200 ⁇ l of Nelson's reagent (aqueous solution of 5.5% of ammonium molybdate, 4.6% of concentrated sulphuric acid and 12% of Na 2 SO 4 .7H 2 O).
  • Nelson's reagent aqueous solution of 5.5% of ammonium molybdate, 4.6% of concentrated sulphuric acid and 12% of Na 2 SO 4 .7H 2 O.
  • the volume of the mixture is adjusted to 5 ml with distilled water and its absorbance is measured by spectrophotometry at a wavelength of 500 nm.
  • the amount of reducing sugars liberated is estimated by comparison with the absorbance values obtained with a calibration series established using a glucose solution.
  • the ⁇ -1,3-glucanase enzymatic activity is expressed in nanomoles of glucose equivalent liberated per minute under the conditions of the enzymatic test described above.
  • the different forms of the recombinant protein were isolated and purified from the centrifugate obtained in section 7 (end of the first paragraph).
  • the isolation and purification comprise steps of extraction, ammonium sulphate precipitation, FPLC liquid chromatography on a cation exchange column based on synthetic polymer and exclusion chromatography (molecular sieving) on a crosslinked agarose, according to the protocol described below:
  • the bacterial pellet is treated with agitation at 4° C. with a pH 8 buffer solution (30 mM Tris, 1 mM EDTA). After centrifugation (15,000 g, 30 min), the supernatant collected (lysate) constitutes the crude extract.
  • the protein extract is precipitated with ammonium sulphate (60% saturation).
  • the proteins which have precipitated are collected by centrifugation (15,000 g for 30 min), solubilised in a buffer solution (100 mM ammonium acetate, pH 5.2) and dialysed overnight at 4° C. against a 100 mM ammonium acetate buffer solution, pH 5.2.
  • the concentration of the buffer solution of the protein extract is brought to 10 mM by passage through ready-to-use minicolumns (Pharmacia PD-10).
  • the protein extract is then purified by ion exchange chromatography on a column based on synthetic polymer (Pharmacia Mono-S column) according to the Pharmacia FPLC technique.
  • the extract is applied to the Mono-S column equilibrated with a 10 mM ammonium acetate buffer, pH 5.2.
  • the proteins retained on the column are eluted with a linear gradient from 10 to 500 mM ammonium acetate.
  • fractions containing a ⁇ - 1,3-glucanase activity are concentrated by ultrafiltration on a Centricon 10 concentration cartridge (Amicron).
  • the purification of the protein is continued by chromatography (molecular sieving) on a crosslinked agarose (Pharmacia Superose 12 column). Elution is carried out with a 500 mM ammonium acetate buffer, pH 5.2.
  • the different forms of recombinant ⁇ -1,3-glucanase are identified by their molecular weight (polyacrylamide gel electrophoresis in the presence of SDS--visualisation with silver), by their activities measured by the colorimetric method described above and by their positive reactions with the polyclonal antibodies directed towards tobacco ⁇ -1,3-glucanase prepared in section 1.
  • sequences correspond to the sequence deduced from the coding portion of plasmid pBR59 (see FIGS. 4A-4C). There is hence no post-translational maturation of the amino-terminal portion. The observed differences in molecular weight for a similar specific activity are hence probably the result of a carboxy-terminal post-translational maturation which occurs on 27 amino acids approximately.
  • Section 9 Construction of a vector for expression in plant cells: the binary vector pBR60.
  • the DNA carrying the coding sequence was obtained by cleavage of the vector M13BR140, obtained in section 6c) (see FIGS. 3A-D and SEQ NO:13), using the restriction enzymes BamHI and SacI, and purified by electrophoresis on low-melting-temperature agarose gel.
  • This DNA was inserted between a promoter sequence comprising so-called 35S promoter of the cauliflower mosaic virus (35S CaMV) and a terminator sequence comprising the nopaline synthase terminator of Agrobacterium tumefaciens.
  • the approximately 900-bp HindIII-BamHI fragment containing the 35S promoter is isolated from plasmid pBI121 (Clontech) by cleavage using the endonucleases HindIII and BamHI followed by agarose gel electrophoresis. This fragment is cut again with HindII.
  • the approximately 410-bp fragment carrying the BamHI site is treated with T4 DNA ligase in the presence of a HindIII linker (synthetic sequence containing a HindIII site). After cleavage with the endonuclease HindIII and agarose gel electrophoresis, the resulting HindIII-BamHI fragment (of approximately 420-bp) is isolated and purified.
  • the promoter sequence, the coding sequence of the complementary DNA of ⁇ -1,3-glucanase and the terminator sequence were ligated using T4 DNA ligase in the binary vector pBIN19 opened using the endonucleases HindIII and EcoRI.
  • This vector carries two genes for resistance to kanamycin, one capable of being expressed in bacteria, and the other, located immediately upstream of the complete recombinant gene (see Bevan, 984, Nucl. Ac. Res., 12, 8711-8721), capable of being transferred to plant cells.
  • the gene for resistance to kanamycin will serve as a selection marker during the steps of transformation and analysis of the progeny of the transformed plants.
  • the vector obtained is referred to as plasmid pBR60.
  • the nucleotide sequence of the complete recombinant gene, verified by sequencing, together with the peptide sequence deduced are shown in FIGS. 5A-5E.
  • the plasmid is cloned into E. coli strain MC1061 (Clontech).
  • Section 1 Transfer into Agrobacterium tumefaciens of plasmid pBR60 containing the recombinant gene for ⁇ -1,3-glucanase.
  • This transfer is carried out as described below by three-way conjugation between the E. coli strain MC1061 containing the vector pBR60 and Agrobacterium tumefaciens strain LBA4404 (Clontech) using E. coli strain HB101 containing the mobilising plasmid pRK2013 (D. M. Figurski et al., 1979, Pro. Ntl. Ac. Sci. USA, 76, 1648-1652).
  • the E. coli strain MC1061 containing plasmid pBR60 and an E. coli strain HB101 (Clontech) containing the mobilising plasmid pRK2013 are cultured at 37° C. in Luria medium (Gibco) in the presence of 25 mg/l of kanamycin.
  • Agrobacterium tumefaciens strain LBA4404 is cultured at 28° C. in Luria medium (Gibco) in the presence of 100 mg/l of rifampicin (it is resistant to this antibiotic); 200 ⁇ l of each of the three cultures are mixed, plated out on Luria agar medium (Gibco) and incubated overnight at 28° C.
  • the bacteria are then resuspended in 5 ml of Luria medium and aliquot portions are plated out on Petri dishes containing an agar minimum medium (described in 'Plant Molecular Biology Manual” Gelvin et al., Kluwer Academic Press, 1988) in the presence of 100 mg/l of rifampicin and 25 mg/l of kanamycin. Under these conditions, only Agrobacterium tumefaciens colonies which have integrated plasmid pBR60 grow (E. coli strains cannot grow under these conditions). These colonies contain the recombinant gene for ⁇ -1,3-glucanase in a context permitting its replication.
  • the resistance of the selected colonies to both antibiotics is verified by transplanting them on the same selection medium twice in succession.
  • the presence of the recombinant gene for ⁇ -1,3-glucanase in Agrobacterium tumefaciens is verified by the Southern blot method on a total DNA preparation (cell lysis, purification of the DNA by extraction using a phenol/chloroform mixture, according to the protocol described by Gelvin in the work cited above, cleavage of the purified DNA using restriction enzymes, agarose gel electrophoresis, transfer onto a membrane and hybridisation according to techniques well-known to those skilled in the art).
  • Section 11 Obtaining transformed tobacco plants
  • Nicotiana tabacum tobacco cultured in vitro was infected with Agrobacterium tumefaciens containing plasmid pBR60 according to the procedure of Horsch et al., well-known to a person skilled in the art (Horsch R. B. et al., 1985, Science 227, 1229-1231), the main steps of which are described below.
  • Discs of axenic N. tabacum tobacco plants are incubated in a culture of A. tumefaciens harbouring plasmid pBR60.
  • the discs, drained on Whatman paper, were transferred onto culture media in Petri dishes in order to multiply the transformed cells so as to obtain calluses, and then to produce buds in the presence of cefotaxime (500 ⁇ g/ml), which is designed to remove Agrobacterium tumefaciens, and kanamycin (100 ⁇ g/ml).
  • Buds resistant to kanamycin were then transferred onto a medium permitting the induction of roots in the presence of cefotaxime and kanamycin.
  • the plantlets are then transplanted into pots in a substrate composed of peat and compost, and left to grow in a greenhouse. All the transformed plants (R0 generation) which survived the steps of regeneration and acclimatisation in the greenhouse proved morphologically normal and fertile. They were self-fertilised and gave seeds (R1 generation).
  • Section 12 Analysis of the genomic DNA of the transformed tobacco plants (R0 generation) according to the Southern blot technique.
  • the high molecular weight genomic DNA was isolated from mature leaves of transgenic plants of the R0 generation according to the method of extraction using cetyltrimethylammonium bromide and of purification by precipitation, described in the work "Plant Molecular Biology Manual” already cited.
  • Section 13 Demonstration of the expression of soybean ⁇ -1,3-glucanase in transformed tobacco plants (of RO generation).
  • Crude protein extracts were prepared from various plant tissues (root, stem, leaf, and the like). The tissue fragments were frozen in liquid nitrogen, reduced to powder and stored at -20° C. The powder was extracted at 4° C. in the presence of a 0.1M ammonium acetate buffer, pH 5.2, and subjected to a centrifugation at 10,000 g. The total protein concentration was determined on the supernatants, hereinafter referred to as crude protein extracts, following the technique of Bradford (Bradford, M. M., 1976, Anal. Biochem., 72, 248-254).
  • loading buffer consisting of 0.125M Tris-HCl, pH 6.8, 4% SDS, 0,002% bromophenol blue, 20% glycerol, 10% ⁇ -mercaptoethanol (according to the protocol described by Laemmli, U. K. Laemmli, Nature, 227, 1970, 680-685);
  • Immunodetection is carried out according to a protocol which comprises the following steps:
  • the antigen-antibody complex is then visualised using an alkaline phosphatase-conjugated streptavidin/biotin system with the Amersham kit RPN 23 ("Blotting detection kit”), used according to the manufacturer's directions.
  • the blot obtained shows the presence of a protein of approximately 37 ⁇ 3 kDa for the transformed plants which is absent from the control plants (the protein deduced from cDNA sequence, from which its assumed signal peptide for an expression in eukaryotic cells has been cleaved, has a molecular weight of 38,156 Da).
  • the apparent molecular weight obtained for the recombinant protein expressed in the transformed tobacco plants was compared with that obtained for the natural protein present in an extract of soybean cells (see section 1) and natural tobacco ⁇ -1,3-glucanase present in an extract of tobacco cells (see section 1), by electrophoretic migration on parallel lanes according to the protocol described by Laemmli (U. K. Laemmli, Nature 227, 1970, 680-685).
  • the sizes of these proteins were compared with those of proteins of known molecular masses (molecular weight markers of between 14,000 and 97,400 Da of Bio-Rad--ref. 61-0304).
  • soybean ⁇ -1,3-glucanase synthesised in tobacco possesses the same apparent molecular weight as that of the natural soybean protein: 37 ⁇ 3 kDa.
  • the post-translational maturation of this protein is hence performed in the same manner in a plant other than soybean.
  • This apparent molecular weight of soybean ⁇ -1,3-glucanase is approximately 3 kDa higher than that of tobacco ⁇ -1,3-glucanase, whose molecular weight is 34,969 Da (H. Shinshi et al., 1988, Proc. Natl. Aca. Sci. USA, 85, 5541-5545).
  • the molecular weight of soybean ⁇ -1,3-glucanase is hence in the region of 38 kDa, which is very close to the molecular weight predicted in section 5 for the mature protein of 338 amino acids predicted in section 5, which is 38,156 Da.
  • Section.14 Measurement of the resistance of transformed formed tobacco plants (R1 generation) to pathogenic fungi.
  • Tobacco plants regenerated (R0 generation) in the presence of kanamycin were self-pollinated.
  • the mature seeds (R1 generation) are sown on Murashige and Skoog medium supplemented with 100 ⁇ g/ml of kanamycin.
  • the kanamycin-resistant plantlets of the R1 generation derived from 13 chosen transformed plants, from a Nicotiana tabacum var. Wisconsin Havana 38 plant sensitive to the fungus Chalara elegans (also known as Thielaviopsis basicola), abbreviated to W38, were transferred to the greenhouse for evaluation of their resistance to this fungus. The latter was chosen since it is representative of the pathogenic fungi of tobacco possessing ⁇ -1,3-glucans in their wall. The study covered numbers of plantlets varying from 16 to 25 depending on the progeny of the transformed plants. The protocol chosen in this study is described below:
  • the plantlets are cultivated in pots (3 ⁇ 3 cm). On appearance of the 5th leaf, the plants are inoculated by depositing a suspension of 5 ⁇ 10 5 endoconidia of this fungus on the neck.
  • the endoconidia are taken from mycelial cultures of this fungus maintained on potato dextrose agar medium (Difco) at 22° C. and in the dark.
  • the resistance to Chalara elegans is evaluated 45 days after inoculation.
  • the plants are scored according to the symptoms of infection and according to their level of vegetative development.
  • Score 3 development of the plants attaining 50% of the development of the control, root system displaying healthy portions
  • the resistance index of the progeny of a transformed plant represents the mean of the scores assigned to the plantlets derived from this plant.
  • Section 15 Purification of the recombinant ⁇ -1,3-glucanase from the leaves of transformed tobacco plants (R1 generation) and determination of its amino-terminal sequence.
  • the recombinant protein was purified from crude extracts of transformed tobacco leaf proteins, by ammonium sulphate precipitation and FPLC liquid chromatography on a cation exchange column based on synthetic polymer on a crosslinked agarose, according to the protocol described below:
  • STEP 1 The protein extract is precipitated with ammonium sulphate (60% saturation). The proteins which have precipitated are recovered by centrifugation (15,000 g for 30 min), solubilised in a buffer solution (100 mM ammonium acetate, pH 5.2) and dialysed overnight at 4° C. against a 100 mM ammonium acetate buffer solution, pH 5.2.
  • the concentration of the buffer solution of the protein extract is brought to 10 mM by passage through ready-to-use minicolumns (Pharmacia PD-10).
  • STEP 2 The protein extract is then purified by ion exchange chromotography based on synthetic polymer (Pharmacia Mono-S column) using an FPLC technique (Pharmacia).
  • the extract is applied to the Mono-S column equilibrated with a 10 mM ammonium acetate buffer, pH 5.2.
  • the proteins retained on the column are eluted with a linear gradient from 10 to 500 mM ammonium acetate.
  • the soybean ⁇ -1,3-glucanase is identified by its molecular weight (polyacrylamide gel electrophoresis in the presence of SDS--visualisation with silver), and by its immunoblot (see Section 13b)) and its activity, measured by the colorimetric method described in Section 8 1).
  • the filter is introduced into a protein sequencer (model 470 A marketed by the company Applied Biosystems (USA)) equiped with a chromatograph (Applied Biosystems model 430) which continuously analyses the phenylthiohydantion derivatives formed, after each degradation cycle.
  • amino-terminal sequence of the recombinant ⁇ -1,3-glucanase begins with a Gln, as predicted by comparison of the already determined amino-terminal sequences of dicotyledons (see section 5).
  • Section 16 Obtaining transformed rape plants.
  • Stem segments are removed from the apical tip of rape plants (Brassica napus: spring varieties Brutor and Westar and winter variety) approximately 1 m in height. These segments are surface sterilised, rinsed in sterile water, cut into segments 1.5 cm in length approximately and placed in a tube containing medium A.
  • Inoculation of the tip of this segment is performed by application of a suspension of the Agrobacterium rhizogenes strain containing plasmid pBR60.
  • Transformed roots appear on the stem segment after 1 to 2 weeks; they are removed and placed on agar medium B (15 g/l) supplemented with 500 ⁇ g of cefotaxime/ml.
  • Root fragments are incubated for 15 days on medium D containing 3 mg/l of 2,4-dichlorophenoxyacetic acid, and then placed on RCC medium for induction of buds. Rooted plants are then obtained by passage of the buds through media F and G.
  • Section 17 Demonstration of the expression of soybean ⁇ -1,3-glucanase in transformed rape plants.
  • Crude protein extracts were prepared from leaves of the plant. These extracts were frozen in liquid nitrogen, reduced to powder and stored at -20° C. The powder was extracted at 4° C. in the presence of a 0.4M ammonium acetate buffer, pH 5.2, and subjected to a centrifugation at 10,000 g. The total protein concentration was determined on the supernatants, hereinafter referred to as crude protein extracts, following the technique of Bradford (Bradford, M. M., 1976, Anal. Biochem., 72,248-254).
  • the crude protein extracts of different transformed plants and of untransformed (control) plants are subjected to a Western blot, a technique well-known to a person skilled in the art and described above.
  • the antigen-antibody complex is then visualised using an alkaline phosphatase-conjugated streptavidin/biotin system with Amersham kit RPN 23 ("Blotting detection kit”), used according to the manufacturer's directions.
  • the blot obtained shows the presence of a protein of approximately 37 ⁇ 3 kDa for the transformed plants, which is absent from the control plants.

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Abstract

The invention provides a protein, isolated from soybean, having β-1,3-glucanase activity and comprising the amino acid sequence of SEQ ID No: 1. DNA constructs encoding said protein, and transformed plants expressing the protein, are also provided.

Description

BACKGROUND OF THE INVENTION
The invention relates to a recombinant DNA coding for a novel protein having β-1,3-glucanase activity, or for a precursor of this protein, to a bacterium containing this recombinant DNA, to a plant cell, plant or plant part, especially a plant seed, transformed by this recombinant DNA, as well as to this novel protein and to a method for preparing it.
Crop plants are known to be subjected to attack by-parasites such as phytopathogenic fungi, which are responsible for substantial harvest losses. The .main means at present for controlling these fungi lies in the use of chemical substances having fungicidal activity. It is now known that plants react naturally to such attack by various defence mechanisms, which are unfortunately, in general, triggered too late and at too low an intensity to be sufficiently effective.
One of these mechanisms comprises the induction of an enzyme known as β-1,3-glucanase E.C.3.2.1.39 (Kombrink et al., 1988, Pr. Ntl. Acad. Sci. USA, 85,982-986 and Pegg et al., 1981, Physiol. Plant. Pathol. 19,371-382). This induction can be triggered artificially through the effect of elicitors, that is to say compounds of biological origin capable of inducing in a healthy plant the defence reactions which it deploys naturally during an infection by pathogenic agents, or during a hormonal imbalance caused by auxin, cytokinins or ethylene (Abeles et al., 1971, Plant Physiol. 47,129-134 and de Loose et al., 1988, Gene, 70,13-23).
β-1,3-glucans are linear polysaccharide polymers consisting of glucose units linked via β-(1→3) linkages, sometimes possessing β-(1→4) or β-(1→6) type branching (Farka 1982, in "Fungal protoplasts", Peberdy and Ferencry, published by Dekker Inc.). These polysaccharides constitute a typical component of the skeleton of the wall of most fungi, and in particular phytopathogenic fungi. β-1,3-glucanases are capable of degrading them by fragmentation of the β-1,3-glucan chains. Most known plant β-1,3-glucanases are of the endo type.
It is known, moreover, that recent progress in so-called recombinant DNA technology and in the transformation of plant cells, as well as in the regeneration of whole plants from the latter, enable a gene of interest to be introduced into plant cells, a plant or a plant part so as to obtain an advantageous phenotype.
SUMMARY OF THE INVENTION
DNA sequences coding for several plant β-1,3-glucanases, in particular a Nicotiana plumbaginifolia β-1,3-glucanase (De Loose et al., 1988, Gene, 70,13-23) and a soybean β-1,3-glucanase (Takeuchi et al., 1990, Plant Physiol., 93,673-682), have been isolated, cloned and determined. Patent Application EP-A-0,353,191 describes the isolation and cloning of different fragments of complementary DNA, the assembling of which enables the complementary DNA sequence coding for a tobacco β-1,3-glucanase to be deduced, as well as of the genomic DNA sequence coding for this enzyme. The document EP-0,392,225 discloses, in particular, the construction of a chimeric gene coding for this tobacco β-1,3-glucanase, the transformation of tobacco with the latter and the verification, by Western blot and visualisation using polyclonal antibodies, of the over-expression of this endogenous protein in the transformed plants. This patent application does not show that the recombinant tobacco β-1,3-glucanase is biologically active, nor a fortiori that it confers a resistance of the said transformed plants to pathogenic agents.
It is known, moreover, that β-1,3-glucanases such as, for example, Bacillus subtilis β-1,3-glucanase are useful enzymes in the conversion of biomass, in particular in some sectors of the papermaking industry and in the agri-foodstuffs industry, especially brewing (Meaden, 1986, Brewers Guardian, 115, 7 and MacQueen, October 1987, New Scientist, 66).
It is known, lastly, that many recombinant proteins can be produced by eukaryotic cells and bacteria, following introduction into these of genes coding for the said proteins using conventional genetic engineering techniques.
The present invention relates to a novel recombinant DNA, characterised in that it codes for a protein having β-1,3-glucanase activity or for a precursor thereof, this protein having β-1,3-glucanase activity comprising the following amino acid sequence (a1) (SEQ ID NO:1):
______________________________________                                    
Ile Gly Val Cys Tyr Gly Met Leu Gly Asn Asn Leu Pro Ser                   
Ala Asn Asp Val Ile Gly Leu Tyr Arg Ser Asn Asn Ile Lys                   
Arg Met Arg Leu Tyr Asp Pro Asn Gln Ala Ala Leu Glu Ala                   
Leu Arg Asn Ser Gly Ile Glu Leu Ile Leu Gly Val Pro Asn                   
Ser Asp Leu Gln Gly Leu Ala Thr Asn Pro Asp Thr Ser Arg                   
Gln Trp Val Gln Lys Asn Val Leu Asn Phe Trp Pro Ser Val                   
Lys Ile Lys Tyr Val Ala Val Gly Asn Glu Val Ser Pro Val                   
Gly Gly Ser Ser Ser Val Ala Gln Tyr Val Leu Pro Ala Ile                   
Gln Asn Val Tyr Gln Ala Ile Arg Ala Gln Gly Leu His Asp                   
Gln Ile Lys Val Ser Thr Ser Ile Asp Met Thr Leu Ile Gly                   
Asn Ser Phe Pro Pro Ser Gln Gly Ser Phe Arg Gly Asp Val                   
Arg Ser Tyr Leu Asp Pro Ile Ile Gly Tyr Leu Val Tyr Ala                   
Asn Ala Pro Leu Leu Val Asn Val Tyr Pro Tyr Phe Ser Tyr                   
Thr Gly Asn Pro Arg Asp Ile Ser Leu Pro Tyr Ala Leu Phe                   
Thr Ala Pro Asn Val Val Val Trp Asp Gly Gln Tyr Gly Tyr                   
Gln Asn Leu Phe Asp Ala Met Leu Asp Ser Val His Ala Ala                   
Ile Asp Asn Thr Lys Ile Gly Tyr Val Glu Val Val Val Ser                   
Glu Ser Gly Trp Pro Ser Asp Gly Gly Phe Ala Ala Thr Tyr                   
Asp Asn Ala Arg Val Tyr Leu Asp Asn Leu Val Arg Arg Ala                   
Asn Arg Gly Ser Pro Arg Arg Pro Ser Lys Pro Thr Glu Thr                   
Tyr Ile Phe Ala Met Phe Asp Glu Asn Gln Lys Asn Pro Glu                   
Ile Glu Lys His Phe Gly Leu Phe Asn Pro Asn Lys Gln Lys                   
Lys                                                                       
______________________________________
or a sequence possessing a high degree of homology with the sequence (a1), (SEQ ID NO:1).
A high degree of homology means here a homology (ratio of identical amino acids to the total number of amino acids) of at least 80%, and preferably at least 90%, of the amino acid sequences when they are aligned on the basis of maximal homology according to the optimal sequence alignment method of Needleman and Wunsch, 1970, J. Mol. Biol., 48, 443-453. This method is used, in particular, in the University of Wisconsin's UWGCG software : Devereux et al., 1984, Nucl. Ac. Res., 12, 8711-8721--GAP option.
The already known peptide sequence closest to that of the sequence (a1) (SEQ ID NO.1): of 307 amino acids is that of the protein of 369 amino acids deduced from the complementary DNA of a Nicotiana plumbaginifolia β-1,3-glucanase SEQ ID NO.12) (see Swissprot data bank ref. Gus$ Nipl. and De Loose et al., 1988, Gene, 70, 13-23). A comparison of these two sequences using the method of Needleman and Wunsch, 1970, J. Mol. Biol., 48,443-453, shows that 213 amino acids out of 307 are identical, equivalent to an approximately 69% homology. This algorithmic method, which considers all possible alignments and creates an alignment, shown in FIGS. 7A and 7B in which the largest possible number of identical amino acids are paired and the number of holes in the aligned sequences is minimal, is used, in particular, in the University of Wisconsin's UWGCG software: Devereux et al., 1984, Nucl. Ac. Res., 12, 8711-8721--GAP option.
This novel recombinant DNA may be used for the expression of this protein having β-1,3-glucanase activity, either to confer an enhanced resistance to pathogenic agents on a plant or plant part expressing the protein, or to produce this protein using eukaryotic cells, in particular ascomycetes such as yeast or filamentous fungi, for example Cryphonectria parasitica, or plant cells, or prokaryotic microorganisms such as, for example, Escherichia coli.
This recombinant DNA can contain, immediately downstream of the nucleotide sequence coding for the amino acid sequence (a1) (SEQ ID NO1), the nucleotide sequence coding for the following amino acid sequence (a4) (SEQ ID NO:4):
______________________________________                                    
Tyr Pro Phe Gly Phe Gly Gly Lys Arg Leu Gly Lys Val Val                   
Ile Asp Asp Phe Asn Ala Thr Thr Ser Ile Lys Ser Asp Val                   
______________________________________
truncated in its carboxy-terminal portion by 0 to 27 amino acids.
Advantageously, this recombinant DNA comprises immediately upstream of the nucleotide sequence coding for the amino acid sequence (a1) (SEQ ID NO:1), a codon for Gln.
An especially high valued DNA such as defined above is that which codes for a protein having β-1,3-glucanase activity or a precursor thereof, and which comprises the following sequence (a5) (SEQ ID NO:3):
______________________________________                                    
Gln                                                                       
Ile Gly Val Cys Tyr Gly Met Leu Gly Asn Asn Leu Pro Ser                   
Ala Asn Asp Val Ile Gly Leu Tyr Arg Ser Asn Asn Ile Lys                   
Arg Met Arg Leu Tyr Asp Pro Asn Gln Ala Ala Leu Glu Ala                   
Leu Arg Asn Ser Gly Ile Glu Leu Ile Leu Gly Val Pro Asn                   
Ser Asp Leu Gln Gly Leu Ala Thr Asn Pro Asp Thr Ser Arg                   
Gln Trp Val Gln Lys Asn Val Leu Asn Phe Trp Pro Ser Val                   
Lys Ile Lys Tyr Val Ala Val Gly Asn Glu Val Ser Pro Val                   
Gly Gly Ser Ser Ser Val Ala Gln Tyr Val Leu Pro Ala Ile                   
Gln Asn Val Tyr Gln Ala Ile Arg Ala Gln Gly Leu His Asp                   
Gln Ile Lys Val Ser Thr Ser Ile Asp Met Thr Leu Ile Gly                   
Asn Ser Phe Pro Pro Ser Gln Gly Ser Phe Arg Gly Asp Val                   
Arg Ser Tyr Leu Asp Pro Ile Ile Gly Tyr Leu Val Tyr Ala                   
Asn Ala Pro Leu Leu Val Asn Val Tyr Pro Tyr Phe Ser Tyr                   
Thr Gly Asn Pro Arg Asp Ile Ser Leu Pro Tyr Ala Leu Phe                   
Thr Ala Pro Asn Val Val Val Trp Asp Gly Gln Tyr Gly Tyr                   
Gln Asn Leu Phe Asp Ala Met Leu Asp Ser Val His Ala Ala                   
Ile Asp Asn Thr Lys Ile Gly Tyr Val Glu Val Val Val Ser                   
Glu Ser Gly Trp Pro Ser Asp Gly Gly Phe Ala Ala Thr Tyr                   
Asp Asn Ala Arg Val Tyr Leu Asp Asn Leu Val Arg Arg Ala                   
Asn Arg Gly Ser Pro Arg Arg Pro Ser Lys Pro Thr Glu Thr                   
Tyr Ile Phe Ala Met Phe Asp Glu Asn Gln Lys Asn Pro Glu                   
Ile Glu Lys His Phe Gly Leu Phe Asn Pro Asn Lys Gln Lys                   
Lys Tyr Pro Phe Gly Phe Gly Gly Lys Arg Leu Gly Lys Val                   
Val Ile Asp Asp Phe Asn Ala Thr Thr Ser Ile Lys Ser Asp                   
Val                                                                       
______________________________________
This recombinant DNA preferably contains, upstream of the sequence coding for the sequence (a1) (SEQ ID NO:1) optionally preceded by a codon for Gln, a signal sequence, chosen in accordance with the host cell, which has the function of enabling the protein to be exported from the cytoplasm.
For an expression in prokaryotic microorganisms such as, for example, Escherichia coli, this signal sequence can be either a sequence derived from a sequence coding for a natural precursor of a protein exported by a prokaryotic microorganism, for example the OmpA signal peptide (Ghrayeb et al., 1984, EMBO Journal, 3, 2437-2442) or an alkaline phosphatase precursor (J. Bact. 1983, 154, 366-3747), or a non-endogenous sequence originating from a sequence coding for a eukaryotic precursor (for example the signal peptide of one of the natural precursors of human growth hormone), or a sequence coding for a synthetic signal peptide (for example that described in French Patent Application No. 2,636,643, of sequence (SEQ ID NO:4):
______________________________________                                    
Met Ala Pro Ser Gly Lys Ser Thr Leu Leu Leu Leu Phe Leu                   
Leu Leu Cys Leu Pro Ser Trp Asn Ala Gly Ala.                              
______________________________________
For an expression in eukaryotic cells such as ascomycetes, for example Saccharomyces cerevisiae yeast or the filamentous fungus Cryphonectria parasitica, this signal sequence is preferably a sequence derived from a sequence coding for a natural precursor of a protein secreted by these cells, for example, for yeast, the invertase precursor (Patent Application EP-0,123,298) or the precursor of the prepro sequence of the alpha pheromone (Patent Application DK 2484/84), or for Cryphonectria parasitica, the precursor of the prepro sequence of endothiapepsin (described in French patent application no. 2 666 590), of sequence (SEQ ID NO: 5):
______________________________________                                    
Met Ser Ser Pro Leu Lys Asn Ala Leu Val Thr Ala Met Leu                   
Ala Gly Gly Ala Leu Ser Ser Pro Thr Lys Gln His Val Gly                   
Ile Pro Val Asn Ala Ser Pro Glu Val Gly Pro Gly Lys Tyr                   
Ser Phe Lys Gln Val Arg Asn Pro Asn Tyr Lys Phe Asn Gly                   
Pro Leu Ser Val Lys Lys Thr Tyr Leu Lys Tyr Gly Val Pro                   
Ile Pro Ala Trp Leu Glu Asp Ala Val Gln Asn Ser Thr Ser                   
Gly Leu Ala Glu Arg                                                       
______________________________________
For an expression in plant cells, either a sequence coding for the signal peptide of a plant cell protein known to be exported, for example the following sequence described in French patent application no. 2 665 177 (SEQ ID NO:6):
______________________________________                                    
Met Arg Arg Thr Ser Lys Leu Thr Thr Phe Ser Leu                           
Leu Phe Ser Leu Val Leu Leu Ser Ala Ala Leu Ala                           
______________________________________
or a signal sequence coding for the signal peptide of the following sequence (a2) (SEQ ID NO:7):
______________________________________                                    
Met Pro Ser Leu Phe Ala Arg Asn Gln Arg Phe Ser Leu                       
Ala Thr Leu Leu Leu Leu Leu Glu Leu Leu Thr Gly Asn Leu                   
Arg Met Ala Asp Ala                                                       
______________________________________
is used as a signal sequence.
The amino acid sequences (a1), (a2) and (a4), (SEQ ID NOS:1, 7 and 2, respectively) can, for example, be encoded by the nucleotide sequences (Na1) (SEQ ID NO:8), (Na2) and (Na4) below:
__________________________________________________________________________
(Na.sub.1):                                                               
ATTGGTGTGT GTTATGGCAT GCTGGGCAAC AATCTACCGT CAGCAAACGA                    
TGTTATAGGT CTTTATAGAT CAAATAACAT AAAGAGAATG AGACTCTATG                    
ATCCTAATCA AGCTGCTCTA GAAGCACTTA GAAATTCTGG CATTGAACTC                    
ATTCTTGGGG TGCCAAACTC TGACCTTCAA GGCCTTGCCA CCAATCCTGA                    
CACTTCTCGT CAATGGGTGC AAAAAAACGT GTTGAACTTT TGGCCTAGTG                    
TCAAAATCAA GTACGTGGCA GTTGGAAATG AAGTGAGTCC CGTTGGAGGC                    
TCTTCTTCGG TAGCCCAATA TGTTCTACCT GCCATCCAAA ATGTATACCG                    
AGCAATAAGA GCTCAAGGCC TTCATGATCA AATCAAGGTT TCAACATCTA                    
TTGACATGAC CCTAATAGGA AACTCTTTCC CTCCATCGCA AGGTTCCTTC                    
AGGGGTGATG TGAGATCATA CCTAGATCCC ATAATTGGGT ACTTGGTATA                    
TGCAAATGCA CCATTACTAG TCAATGTGTA CCCTTATTTT AGTTACACTG                    
GTAACCCCCG TGACATATCA CTTCCCTATG CTCTTTTCAC AGCACCAAAT                    
GTTGTGGTAT GGGATGGTCA ATATGGGTAC CAAAATTTGT TTGATGCTAT                    
GTTGGATTCA GTACATGCAG CCATTGATAA CACTAAGATT GGTTATGTGG                    
AGGTTGTTGT ATCCGAGAGT GGGTGGCCAT CAGATGGAGG ATTTGCTGCC                    
ACTTATGACA ACGCACGCGT GTACTTAGAC AATTTGGTTC GTCGTGCTAA                    
TAGAGGAAGC CCAAGAAGGC CTTCGAAGCC CACTGAGACT TATATATTTG                    
CCATGTTCGA TGAAAATCAA AAAAATCCAG AGATAGAGAA ACATTTTGGG                    
CTCTTCAATC CCAACAAACA AAAAAAA                                             
(Na.sub.2) (SEQ ID NO: 9):                                                
ATGCCTTCTC TCTTCGCTAG AAACCAGAGG TTCTCATTGG CTACTC.TCCT                   
GCTTCTTCTG GAACTATTGA CAGGAAACCT TCGCATGGCA GATGCT                        
(Na.sub.4) (SEQ ID NO: 10):                                               
TACCCATTTG GGTTTGGAGG AAAGAGGCTA GGGAAAGTTG TTATTGACGA                    
CTTCAATGCA ACAACTTCCA TTAAGAGTGA TGTG                                     
__________________________________________________________________________
The invention also relates to a unit for expression of the recombinant DNA defined above, advantageously carried out by a vector, termed expression vector.
For an expression in procaryotic microorganisms, especially in Escherichia coli, the recombinant DNA must be inserted in an expression unit containing, in particular, an effective promoter, followed by a ribosome binding site upstream of the gene to be expressed, as well as an effective transcription stop sequence downstream of the gene to be expressed. This unit must also contain a selection marker or be introduced into the host cell at the same time as a unit for expression of a selection marker (for example using an expression vector which carries both of these units). All these sequences must be chosen in accordance with the host cell.
For an expression in eukaryotic cells such as ascomycetes, the expression unit according to the invention comprises the recombinant DNA defined above with the means needed for its expression.
For an expression in ascomycete cells such as yeast, for example Saccharomyces cerevisiae, it is appropriate to insert the recombinant DNA between, on the one hand sequences recognised as an effective promoter, and on the other hand a transcription terminator. The expression unit carries a selection marker or is introduced into the host cell at the same time as a selection marker. Preferably, this selection marker is an auxotrophic marker (which complements a mutation of the recipient cells) which permits the selection of cells which have integrated the recombinant DNA in a high copy number, either into their genome or into a multicopy vector. For an expression in ascomycete cells such as those of filamentous fungi, for example those of the genera Aspergillus, Neurospora, Podospora, Trichoderma or Cryphonectria, the expression unit according to the invention carries the recombinant DNA defined above with the means needed for its expression, and optionally a selection marker and/or telomeric sequences. It is, in effect, possible to select transformants which have integrated a DNA of interest using a selection marker located either on the same unit as the DNA of interest or on another unit, these two units then being introduced by cotransformation. The recombinant DNA of the invention may be either integrated in the genome of the filamentous fungi, or preserved in extrachromosomal form by means of sequences enabling this DNA to be replicated and split.
For an expression in plant cells, it is appropriate to insert the recombinant DNA defined above between a promoter and a terminator which are effective in plants.
The promoter is preferably a strong constitutive promoter, for example the 35S promoter of the cauliflower mosaic virus, or a promoter controlling the tissue- or organ-specific expression, such as the promoter of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase, which is expressed preferentially in the leaves and most especially the mesophyll tissues (Kuhlemeier et al., 1987, Ann Rev Plant Physiol 38: 221-257). It is also possible to use a specific promoter controlling, for example, an expression in the seeds or during a precise stage of development of the plant, or a promoter which is inducible following a thermal shock, a wound or interaction between the plant and parasites (Kuhlemeier et al., 1987, reference cited above), if an expression of recombinant DNA is desired in these situations.
The terminator sequence containing polyadenylation sites which can be isolated from plant genes or from genes which are expressed in plants, such as, for example, the nopaline synthase terminator Agrobacterium tumefaciens, is used.
The invention also relates to a bacterium, for example of the species E. coli, which contains the recombinant DNA defined above with the means needed for its replication and its expression. This bacterium may be used in the preparation of a protein having β-1,3-glucanase activity.
The invention also relates to a bacterium, for example of the species E. coli, which contains the recombinant DNA defined above with the means enabling it to be replicated, which bacterium may hence be used for the cloning of this recombinant DNA, and also to a bacterium capable of infecting a plant with transfer of genetic material, for example of one of the species Agrobacterium rhizogenes and Agrobacterium tumefaciens, which contains this DNA in a context enabling it to be replicated and may hence be used for transforming plant cells. The transformation of plant cells with the above recombinant DNA may also be performed by another biological method such as the pollen tube method (Zhong-xun Luo et al., Plant Molec. Biol. Rep., 1988, 6, 165-176) and the direct transformation of germinating seeds (Toepfer R. et al., 1989, The Plant Cell., 1, 133-139), or by a physical method such as the use of polyethylene glycol, electroporation (Chistou P. et al., 1987, Proc. Ntl. Acad. Sci. USA, 84, 3662-3699) and bombardment using microprojectiles (Klein T. M. et al., 1988, Proc. Ntl. Acad. Sci. USA, 85, 8502-8505).
The invention also relates to a plant cell, characterised in that it is transformed by the recombinant DNA defined above with the means needed for its expression. This plant cell can originate from a major crop species such as, for example, maize, soybean, beet, wheat, barley, poppy, rape, sunflower, alfafa and sorghum, a floral species such as rose, carnation and gerbera or a culinary species such as carrot, tomato, lettuce, chicory, pepper, melon and cabbage. Especially highly valued species are Brassica napus rape, Helianthus annuus sunflower and Nicotiana tabacum tobacco.
The step of transformation which involves one or a few cells is followed by a step of multiplication of these transformed cells so as to obtain calluses, which can give rise to transformed plants by processes of organogenesis or embryogenesis.
The invention hence also relates to a plant or plant part, characterised in that it contains the recombinant DNA defined above with the means needed for its expression. An especially highly valued plant part is the part capable of forming a complete new plant, in particular after sowing, burying in the ground or transplanting, or of producing seeds. Such a plant part is, for example, a grain, a mature fertilised ovule, a seed, a cutting, a runner, and the like. These plants can be any one of the above species, and more especially of the species Nicotiana tabacum, Helianthus annuus and Brassica napus.
The invention also relates to a method for obtaining plants resistant to parasites, such as phytopathogenic fungi, which comprises a step of transformation of plant cells with this recombinant DNA, followed by a step of multiplication of the transformed cells and a step of regeneration of the plants.
Preferably, the step of transformation of the plant cells is performed in vitro using an agrobacterium (that is to say a bacterium of the genus Agrobacterium) which has integrated recombinant DNA of interest.
The invention also relates to plants resistant to pathogenic agents, which are capable of being obtained using the method defined above.
The invention also relates to the use of a plant containing the recombinant DNA defined above with the means needed for its expression, as a parent in a selection programme for creating new plant varieties.
The invention also relates to a novel protein having β-1,3-glucanase activity, which comprises the sequence (a1) (SEQ ID NO:1) and optionally, immediately downstream of the sequence (a1), the sequence (a4) (SEQ ID NO:2) truncated in its carboxy-terminal portion by 0 to 27 amino acids, and to a novel protein comprising the sequence (a5) (SEQ ID NO:3).
This protein preferably possesses an apparent molecular mass of 36±3, 37±3 or 39±3 kDa.
This protein is of interest as an enzyme for conversion of biomass which contains β-1,3-glucans, in particular in some sectors of the papermaking industry and in the agri-foodstuffs industry, especially brewing.
The invention also relates to a method for preparing this protein, which comprises the culturing of bacteria, plant cells, plant calluses, plants or plant parts containing the recombinant DNA defined above, lysis thereof and isolation and purification of this protein.
A better understanding of the invention will be gained from the description below, divided into sections, which comprises experimental results and a discussion of the latter. Some of these sections relate to experiments performed with the object of carrying out the invention, and others to examples of embodiment of the invention which are naturally given purely by way of illustration.
A large part of the collective techniques below, which are well known to a person skilled in the art, is described in detail in the work by Sambrook et al.: "Molecular cloning: a Laboratory manual" published in 1989 by Cold Spring Harbor Press editions, New York (2nd edition).
BRIEF DESCRIPTION OF THE DRAWINGS
A better understanding of the description below will be gained by reference to FIGS. 1 to 7.
FIG. 1 shows a restriction map of the HindIII-EcoRI fragment contained in plasmid pBR 1310 and containing the complementary DNA coding for soybean β-1,3-glucanase.
FIGS. 2A-2D show the nucleotide sequence (SEQ ID NO:11) of the HindIII-HindIII insert, part of the complementary DNA of soybean β-1,3-glucanase, the restriction sites used for cloning in M13mp19 and for the subsequent constructions being indicated by arrows, as well as the peptide sequence (SEQ ID NO:12) deduced from this complementary DNA.
FIGS. 3A-3D show the nucleotide sequence (SEQ ID NO:13) of the HindIII-HindIII fragment of the complementary DNA of soybean β-1,3-glucanase after mutagenesis (at the end of section 6c), the restriction sites used for cloning in M13mp19 and for the subsequent constructions being indicated by arrows, as well as the peptide sequence of the protein translated.
FIGS. 4A-4C show the nucleotide sequence (SEQ ID NO:15) of the NdeI-HindIII fragment of the coding portion of plasmid pBR59, an expression plasmid in E. coli, flanked by NdeI and HindIII restriction sites, and the peptide sequence (SEQ ID NO:16): of the protein translated.
FIGS. 5A-5E show the nucleotide sequence (SEQ ID NO:17) of the complete recombinant gene and the peptide sequence of (SEQ ID NO:18) of the protein translated.
FIGS. 6A-6B show the peptide sequence (SEQ ID NO:25) of the common portion of the proteins translated in E, coli and in tobacco.
FIGS. 7A and 7B show an alignment, on the basis of the method of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443-453, of the peptide sequence (SEQ ID NO:12) deduced from the complementary DNA of soybean β-1,3-glucanase (see FIGS. 2A-2D) and the closest known peptide sequence (SEQ ID NO:26), that deduced from the complementary DNA of Nicotiana plumbaginifolia β-1,3-glucanase (Swissprot data bank ref. Gub$-Nipl).
DETAILED DESCRIPTION OF THE INVENTION
Section1: Preparation of polyclonal antibodies against tobacco β-1,3-glucanase
a) Purification of tobacco β-1,3-glucanase.
A tobacco β-1,3-glucanase was purified to homogeneity from tobacco calluses as described below. Tobacco calluses were cultured in vitro on a Murashige and Skoog medium (Murashige T. and Skoog F., 1962, Physiol. Plant., 15, 473-497). Cell extracts are obtained by grinding the plant material in a 50 mM Tris-HCl buffer solution, pH 8.4, containing 15 mM β-mercaptoethanol and 5% of polyvinylpyrrolidone.
The protein is purified from this extract by ammonium sulphate precipitation, liquid chromatography on a cation exchange column based on synthetic polymer and exclusion chromatography (molecular sieving) on a crosslinked agarose, according to the protocol described below.
Step 1:
The protein extract is treated with ammonium sulphate (43% saturation). The proteins which have precipitated are collected by centrifugation (15,000 g for 30 min), solubilised in a buffer solution (100 mM ammonium acetate, pH 5.2) and dialysed overnight at 4° C. against a 100 mM ammonium acetate buffer solution, pH 5.2.
Immediately before proceeding, the acetate concentration of the buffer solution of the protein extract is brought to 10 mM by passage through ready-to-use minicolumns (Pharmacia PD-10).
Step 2:
The protein extract is then purified by ion exchange chromatography using a column based on synthetic polymer (Pharmacia Mono-S column) according to the Pharmacia FPLC technique.
The extract is applied to a Mono-S column equilibrated with a 10 mM ammonium acetate buffer, pH 5.2. The proteins retained on the column are eluted with a linear gradient from 10 to 500 mM ammonium acetate.
At each step, the β-1,3-glucanase is identified by its molecular weight (polyacrylamide gel electrophoresis in the presence Of SDS--visualisation with silver), and its activity is measured by a colorimetric method (see section 8 below) using laminarin (β-1,3-glucan extracted from Laminaria digitataz--Sigma--ref. L9634) as a substrate.
b) Preparation of polyclonal antibodies.
Rabbits were then injected with 25 μg of tobacco β-1,3-glucanase in 500 μl of Freund's complete adjuvant. Three booster injections of 25 μg in Freund's incomplete adjuvant (500 μl) were carried out at 3-week intervals. The immune serum was drawn 3 weeks after the final injection.
This immune serum recognises tobacco β-1,3-glucanase; it also recognises soybean β-1,3-glucanase. It was, in effect, verified that it enables the latter protein to be visualised by the Western blot technique (described in section 13 below) from an extract of total soybean (Glycine max cv Mandarin) proteins.
Section 2: Construction of a phage library of complementary DNA from messenger RNAs of soybean cell cultures.
a) Preparation of messenger RNAs of soybean cells.
The total RNA of 5-day-old soybean cells (Glycine max cv Mandarin), cultured in vitro in the absence of auxin according to the method described by Leguay et al., 1987, Develop. Genetics 8: 351-364, was extracted according to the method described by Jouanneau et al., 1984 Plant Physiol 74; 663-668, summarised below. The cells, washed beforehand in saline solution, are ground in liquid nitrogen; the homogenate is then extracted with a mixture of redistilled phenol and chloroform. After an ethanol precipitation step, the total RNA is dissolved in a solution buffered to pH 7.6.
The poly(A)+ fraction of the messenger RNA (mRNA) was isolated after 2 cycles of affinity chromatography on an oligo(dT)-cellulose column as described in Sambrook et al., ("Molecular cloning: A Laboratory manual", second edition, Cold Spring Harbor Laboratory 1989). Quantification of the messenger RNAs is carried out by spectrophotometry according to a protocol well known to a person skilled in the art.
b) Synthesis of complementary DNAs
Complementary DNAs (cDNAs) were synthesised according to the method described by Gubler and Hoffman, 1983 (Gene, 25: 263-269), which method favours the synthesis and cloning of complete cDNAs: 7 μg of mRNA were treated in this manner for the manufacture of the first strand of cDNA using the Promega "Protoclone GT system" kit (ref. P3010).
c) Cloning of complementary DNAs.
The synthesised double-stranded cDNA is then methylated using EcoRI methylase under the conditions described in the reference work by Sambrook et al. (op. cit.). This enzyme enables the possible EcoRI sites of the cDNA to be protected against cleavage by the endonuclease EcoRI, this protection disappearing for the replicates of the cDNA (which are not methylated).
Synthetic EcoRI linkers (double-stranded DNA fragments containing the EcoRI site) are then added by ligation to the ends of the cDNA. After cleavage with the endonuclease EcoRI and removal of the linkers by chromatography on a column of G-50 molecular sieve (Pharmacia), the cDNA is ligated using T4 DNA ligase in phage lambda gt11, a cloning and expression vector described by Huynh et al., "DNA cloning: a practical approach" IRL Press, D. M. Glover 1985, p. 49, according to the Promega protocol (ref. T3011) "lambda gt11 system", the phage DNA being opened beforehand with the restriction endonuclease EcoRI and dephosphorylated. Aliquot portions of the ligation medium are then packaged in phage particles using the kit marketed by Amersham ("In vitro packaging system for lambda DNA", ref. N334).
The number of recombinants is then estimated by counting the lytic plaques obtained on a bacterial lawn of E. coli strain Y1090 (Sambrook et al., ref. above, op. cit.). Approximately 106 clones were obtained. The plating out of an aliquot portion of the phage suspension on a bacterial lawn in the presence of X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) and isopropyl β-D-thiogalactoside (IPTG), according to the technique described by Huynh et al., ref. above, enabled it to be determined that 81% of these phages integrated a soybean cDNA.
3: Section Immunological screening of the phage library of complementary DNA constructed from soybean cell messenger RNAs.
The production of a library in the vector lambda gt11 makes it possible to carry out the expression of the cloned cDNAs, that is to say to synthesise the proteins encoded by the messenger RNAs which were used for constructing this library. This synthesis takes place after induction with IPTG (isopropyl β-D-thiogalactoside); the synthesised proteins can then be recognised by the antibodies obtained against tobacco β-1,3-glucanase (see section 1). The clones producing these proteins can then be identified and isolated according to a protocol known to a person skilled in the art and described, for example, in Sambrook et al. (op. cit.).
106 phages of the soybean cDNA library are plated out on Petri dishes, and lytic plaques are obtained after 2 h 30 min of incubation at 42° C. A nitrocellulose filter (Schleicher and Schell, BA 85) impregnated with IPTG is placed on the surface of the dishes and left in contact with the agar medium for 4 h at a temperature of 37° C., and then replaced by a second filter which is left for 6 h on the same medium.
The filters removed from the dish are then immersed for 30 min in a solution, referred to as TNT solution, composed of 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% of Tween 20 detergent, and then for 30 minutes in a buffer, referred to as blocking buffer, composed of gelatin in 1% solution in TNT solution. The filters are then treated for 3 h in a solution containing the anti-β-1,3-glucanase polyclonal antibodies obtained above (see Section 1), diluted in the blocking buffer. They were then immersed for 10 minutes successively in each of the following solutions:
TNT solution+0.1% BSA (bovine serum albumin)
TNT solution+0.1% BSA+0.1% Nonidet P40 detergent (Sigma)
TNT solution+0.1% BSA.
The antigen-antibody complex formed is then visualised using secondary antibodies conjugated by coupling with a peroxidase ("Immuno conjugate GAR/IgG (H+L)/Po" of Nordic Immunology, Tilburg, Holland). The chemical visualisation reaction employs 4-chloro-1-naphthol as a chromogenic substrate (giving a blue precipitate). The positive lyric plaques, that is to say those corresponding to clones which synthesise β-1,3-glucanase, are then identified on the Petri dish, and the bacteriophages are removed and purified by means of a secondary immunological screening, performed in a strictly identical manner to the primary screening. One of these clones was selected for continuation of the study.
Section 4: Partial characterisation of the phage DNA of the soybean β-1,3-glucanase clone.
a) Preparation of phage DNA.
The selected phage clone is amplified and its DNA is extracted according to the protocol described in the Amersham kit "cDNA cloning system GT11" (ref. RPN 1280), summarised below. The phages are removed using a coring tool and transferred to a culture of E. coli strain Y1090; after 15 min, 5 ml of Luria medium (Gibco) containing 5 mM of CaCl12 and 50 μg/ml of ampicillin are added. After incubation for 4 h at 43° C. with agitation, the culture is centrifuged at 3,000 rpm for 10 min. The DNA of the phage particles present in the supernatant is then extracted and purified by treatment with polyethylene glycol and sodium chloride NaCl, centrifugation, treatment with proteinase K and precipitation.
b) Analysis of the phage DNA of the selected clones
DNA of the β-1,3-glucanase clones was hydrolysed with the restriction enzyme EcoRI. A single approximately 2,300-bp EcoRI-EcoRI fragment was thereby obtained by cleavage at the cloning sites. There is hence no EcoRI site in the complementary DNA.
Analysis by agarose gel electrophoresis of the fragments obtained by the action of several endonucleases on the approximately 2,300-bp EcoRI-EcoRI fragment enabled it to be shown that the latter contains a PvuII site a few base pairs from one of the EcoRI sites.
Section 5: Construction of plasmid pBR1310, isolation of the complementary DNA insert coding for soybean β-1,3-glucanase and determination of its sequence.
a) Cloning into plasmid pGEM-3Z
By hydrolysis of the clone selected using the restriction enzyme EcoRI and PvuII, the approximately 2,300 bp EcoRI-PvuII fragment containing the soybean β-1,3-glucanase cDNA was isolated after electrophoresis on low-melting-temperature agarose gel.
Plasmid pGEM-3Z (Promega, Madison, Wis, U.S.A.) was opened using the restriction endonucleases EcoRI and SmaI, then purified and isolated by electrophoresis on low-melting-temperature agarose gel. This plasmid comprises a "polylinker" (cloning multisite) successively containing EcoRI, SmaI and HindIII restriction sites.
Ligation using T4 DNA ligase of the approximately 2,300-bp EcoRI-PvuII fragment, containing the cDNA coding for soybean β-1,3-glucanase, into this plasmid thus opened enables a plasmid referred to as plasmid pBR1310 (ligation of the SmaI and PvuII blunt ends with disappearance of these sites) to be obtained, which plasmid is cloned into the E. coli bacterium strain JM109 (Sambrook et al., op. cit.). The vector obtained was then extracted and purified by the alkaline lysis method (Birnboim and Doly, in Sambrook et al., op. cit.).
The use of several restriction enzymes made it possible to draw up the restriction map of the HindIII-EcoRI fragment contained in plasmid pBR1310 and containing the cDNA coding for β-1,3-glucanase, shown in FIG. 1.
The HindIII-HindIII fragment (of approximately 1490 bp) was prepared by the digestion of the HindIII-EcoRI fragment with the endonuclease HindIII, purified by electrophoresis on low-melting-temperature agarose gel and isolated. This fragment was cloned into the DNA of the replicative form of the single-stranded phage M13mp19 (Pharmacia) opened at the HindIII restriction site. The vector M13 containing the 1490-bp HindIII-HindIII insert is referred to as M13BR137. The sequence of this insert is determined according to the dideoxyribonucleotide method (Sanger et al., PNAS-USA, 14, 5463-5467, 1977).
b) Analysis of the cDNA sequence of soybean 62 -1,3-glucanase.
A better understanding of the description below will be gained with the aid of FIGS. 2A-2D.
This sequence contains a single open reading frame (not interrupted by a stop codon) compatible with the apparent molecular weight observed by agarose gel electrophoresis: the sequence beginning with an ATG codon at position 114 and terminating with the TAA stop codon at position 1224-1226 coding for a protein of 370 amino acids.
The University of Wisconsin's UWGCG software: Devereux et al., 1984, Nucl. Ac. Res., 12, 8711-8721-option: Testing for a signal peptide according to the method of G. von Heijne, 1986, Nucl. Ac. Res., 14, 483-490, provides in this sequence for a single portion coding for a signal peptide for an expression in eukaryotic cells, the following sequence (Na2) referred to as a pre sequence (beginning at nucleotide 114 and terminating at nucleotide 209):
__________________________________________________________________________
ATGCCTTCTC TCTTCGCTAG AAACCAGAGG TTCTCATTGG CTACTCTCCT                    
GCTTCTTCTG GAACTATTGA CAGGAAACCT TCGCATGGCA GATGCT                        
__________________________________________________________________________
coding for the signal peptide of 32 amino acids of the following sequence (a2) (SEQ ID NO:7):
______________________________________                                    
Met Pro Ser Leu Phe Ala Arg Asn Gln Arg Phe Ser Leu Ala                   
Thr Leu Leu Leu Leu Leu Glu Leu Leu Thr Gly Asn Leu Arg                   
Met Ala Asp Ala                                                           
______________________________________
A signal peptide is expected by a person skilled in the art, since β-1,3-glucanases are proteins which can be naturally either accumulated in the vacuoles of plant cells or secreted, thereby demanding the presence of a signal peptide.
The already-known peptide sequence closest to that of the protein of 370 amino acids deduced from the complementary DNA of soybean β-1,3-glucanase (SEQ ID NO:12) is that of the protein of 369 amino acids deduced from the complementary DNA of a Nicotiana plumbaginifolia β-1,3-glucanase (SEQ ID NO:22) (see Swissprot data bank ref. Gub$Nipl. and De Loose et al., 1988, Gene, 70, 13-23).
A comparison of these two sequences using the method of Needleman and Wunsch, 1970, J. Mol. Biol, 48,443-453, employed in the University of Wisconsin's UWGCG software: Devereux et al., 1984, Nucl. Ac. Res. 12,8711-8721, GAP option, shows that 224 amino acids are identical, equivalent to an approximately 60% homology. This algorithmic method considers all possible alignments and creates an alignment, shown in FIGS. 7A and 7B, in which the largest possible number of identical amino acids are paired and the number of holes in the aligned sequences is minimal.
Comparison of the amino-terminal region of the predicted mature soybean β-1,3-glucanase with the published amino-terminal sequences of β-1,3-glucanases
Four plant β-1,3-glucanases are known at present, the amino-terminal sequences of which have been determined experimentally. They are the β-1,3-glucanases of Nicotiana tabacum (H. Shinshi et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5541-5545), of Nicotiana plumbaginifolia (M. De Loose et al., 1988, Gene, 70, 13-23), of Phaseolus vulgaris bean (B. V. Edington et al., 1991, Plant Molecular Biology, 16, 81-94) and of Hordeum vulgare barley (P. B. Hoj et al., 1988, Febs Lett., 230, 67-71).
Their amino-terminal sequences display a strong homology, of the order of 80% for the 15 amino-terminal amino acids. In the case of the dicotyledons (Nicotiana tabacum, Nicotiana plumbaginifolia and Phaseolus vulgaris), they all begin with a Gln followed, after a possible Ser, by the sequence Ile Gly Val Cys Tyr Gly (amino acid residues 1-6 of SEQ ID NO:12). It may be noted that the sequence of mature soybean (dicotyledon) β-1,3-glucanase predicted by the above software also begins with the sequence Gln Ile Gly Val Cys Tyr Gly (amino acid residues 1-6 of SEQ ID NO:12).
Section 6: Construction of a vector for expression
in E. coli: plasmid pBR59.
a) Silent mutagenesis of the coding portion to remove the SacI restriction site (at position 575) of the coding portion.
The sequence GAG CTC of the SacI restriction site located at position 575 of the vector M13BR137 is replaced, by directed mutagenesis as described below, by the sequence GAG CAC. The codon GCT, corresponding to an alanine residue in the initial cDNA sequence, is hence replaced by the codon GCA, also corresponding to an alanine residue (silent mutation).
The single-stranded form of the vector M13BR137 is isolated according to the protocol described by Sambrook et al., op. cit., from a culture of the E. coli bacterial strain DH5αF' (Gibco-BRL) transformed beforehand with this vector according to the protocol recommended by the manufacturer. On this single-stranded template, mutagenesis is performed according to the protocol of the "Oligonucleotide-directed in vitro mutagenesis system" kit (Amersham--ref. RPN 1523--version 2), using an oligonucleotide synthesised chemically on the Millipore Biosearch 8700 DNA synthesiser, the sequence of which is shown below (SEQ ID NO:19):
GATCATGAAG GCCTTGTGCT CTTATTGCTT GG
The directed mutagenesis technique described in detail in the booklet accompanying this kit, and summarised below, consists in hybridising the above oligonucleotide with the single-stranded form of the vector M13BR137, and then in reacting Klenow polymerase and T4 DNA ligase in the presence of the thionucleotide dCTP-α-S (α-thiodeoxycytidine triphosphate) so as to obtain a circular double-stranded form of the recombinant phage, one of the strands of which carries the desired mutation and is protected from cleavage by the endonuclease NciI and degradation by exonuclease III. On the other strand (which has served as a template) a break is then introduced using the endonuclease NciI, and this strand is thereafter removed by the subsequent action of exonuclease III.
The resulting vector, referred to as vector M13BR138, was cloned into E. coli strain DH5αGF' and sequenced; it possesses, as expected, a sequence mutated by replacement of a codon GCT by a codon GCA, which has eliminated the SacI restriction site from the coding portion.
b) Mutagenesis of the 5' portion of the insert coding for soybean β-1,3-glucanase to introduce NdeI and BamHI restriction sites.
The single-stranded form of the vector M13BR138 is isolated as described above, and a directed mutagenesis is then carried out under the same conditions as before using the chemically synthesised oligonucleotide the sequence of which is noted below (SEQ ID NO:20)
GAGAAGGCAT GGATCCAAAC ATATGAATAC ACCAC
The vector derived from this mutagenesis, referred to as vector M13BR139, is cloned into the E. coli bacterial strain DH5αF'; it was sequenced, which confirmed the introduction of NdeI and BamHI restriction sites upstream of the ATG translation initiation codon.
c) Mutagenesis of the 3'portion of the insert of the vector M13BR139 to introduce HindIII and SacI restriction sites.
The single-stranded form of the vector M13BR139 is isolated, and a directed mutagenesis is then carried out on the 3'portion of the insert using the oligonucleotide of the following sequence and under the conditions already described SEQ ID NO:21:
CAGAGATTTT GAAGCTTAGG AGCTCAACCT TACACATC
The vector resulting from this mutagenesis, referred to as vector M13BR140, is cloned into the E. coli bacterial strain DH5αF'. Its sequence was determined, which confirmed the creation of HindIII and SacI restriction sites downstream of the TAA stop codon. This sequence is shown in FIGS. 3A-3D and in SEQ ID NO:13.
d) Construction of plasmid pBR141.
The replicative form of the vector M13BR140 is isolated and purified according to the protocol of Birnboim and Doly described in Sambrook et al., op. cit. The NdeI-HindIII insert containing the β-1,3-glucanase coding portion was isolated by digestion with the restriction enzymes NdeI and HindIII, and purified by agarose gel electrophoresis and electroelution (Sambrook et al., op. cit.). This insert is then ligated using T4 DNA ligase in the large NdeI-HindIII fragment of plasmid p373,2, the construction and constitutive elements of which are described in Patent Application EP-A-0,360,641 (FIGS. 3A-3D of this document shows the restriction map of this plasmid), opened beforehand by digestion with the restriction enzymes NdeI and HindIII. This large fragment contains, in particular, successively from the NdeI end to the HindIII end, a promoter analogous to the "tac" promoter (Sambrook et al., op. cit.) which is inducible with isopropyl β-D-galactoside, a gene coding for β-lactamase (conferring resistance to ampicillin) in a context permitting its expression, an origin of replication in E. coli and a transcription terminator derived from the phage fd. The vector obtained, referred to as plasmid pBR141, is cloned into E. coli strain RR1. The structure of the plasmid is verified by producing a restriction map.
e) Construction of plasmid pBR59
The plasmid DNA of the clone containing plasmid pBR141 is extracted according to the protocol of Birnboim and Doly (Sambrook et al., op. cit.).
Removal of the envisaged prokaryotic pre sequence (see section 5) of soybean β-1,3-glucanase is carried out by removal of the sequence comprised between the NdeI and SphI restriction sites and replacement of this sequence by the synthetic linker of sequence given below (SEQ ID NO:22):
TATGATTGGT GTGTGTTATG GCATGACTAA CCACACACAA TACC
so as to re-form the sequence coding for the protein devoid of the signal peptide envisaged in prokaryotic cells, the first amino acid of this protein, namely the glutamine residue (capable of cyclising to pyroglutamate, which makes it impossible to determine the amino-terminal sequence), being replaced by a translation initiation methionine residue.
Ligation of this linker using T4 DNA ligase enables a plasmid, referred to as plasmid pBR59, to be obtained, which plasmid is cloned into the E. coli bacterial strain RR1. In this vector, the β-1,3-glucanase is under the control of a promoter (described in Patent Application EP-A-0,360,641) analogous to the "tac" promoter (Sambrook et al., op. cit.) which is inducible with isopropyl β-D-thiogalactoside (IPTG). The sequence of the coding portion of plasmid pBR59, flanked by the NdeI and HindIII sites, is shown in FIGS. 4A-4C and in SEQ ID NO:15.
Section 7: Expression of soybean β-1,3-glucanase in E. coli.
The E. coli strain RR1 containing the expression vector pBR59 and an E. coli strain RR1 containing plasmid p373,2 are cultured in Luria medium (Gibco) containing 100 mg/1 of the antibiotic ampicillin overnight at 37° C. After a dilution of the culture to 1/100 in the same medium, the bacteria are returned to culture for 1 hour at 37° C. and IPTG is then added at a final concentration of 1 mM. Culturing is then continued for 3 hours and the bacteria are harvested by centrifugation.
The bacteria are resuspended in a buffer, referred to as loading buffer, of the following composition:
0.125M Tris-HCl, pH 6.8
4% sodium dodecyl sulphate
20% glycerol
0.02% bromophenol blue
10% β-mercaptoethanol and the mixture is then brought to 100° C. for 10 min (causing lysis of the bacteria and denaturation of the proteins). 10 μg of solubilised proteins are applied to a polyacrylamide electrophoresis gel according to the protocol described by Laemmli (U. K. Laemmli, Nature 227, 1970, 680-685). After electrophoresis, the gel is stained using Coomassie blue. The presence of three extra bands absent in the case of the control strain is noted in the case of the β-1,3-glucanase clone.
Section 8: Measurement of the enzymatic activity of the recombinant soybean β-1,3-glucanase expressed in E. coli, isolation and purification of three forms of this protein and determination of their amino-terminal sequences.
1. Measurement of the enzymatic activity of the recombinant β-1,3-glucanase.
β-1,3-glucanase activity is measured by a colorimetric method enabling the amount of monomers or oligomers liberated by the enzyme from a substrate (laminarin) to be estimated by determining the reducing power of the sugars thus liberated. This method, described by G. Ashwell, 1957, in "Methods in Enzymology III", 73-105, S. P. Colowick and N. U. Kaplan Eds., is summarised below.
50 μl of a solution containing 50 mg/ml of laminarin (β-1,3-glucan extracted from Laminaria digitata--Sigma--ref. L9634) are added to a solution containing a concentration of β-1,3-glucanase chosen so as to fall within a linear response range. The volume of the reaction mixture is adjusted to 500 μl using a 0.2M sodium acetate buffer solution, pH 5.0. After incubation for one hour at 40° C., a 200 μl aliquot portion is added to 200 μl of Somogyi's reagent (mixture of 25 ml of an aqueous solution comprising 2.5% of Na2 CO3, 2.5% of KNaC4 H4 O6.4H2 O, 2% of NaHCO3, 20% of Na2 SO4 and 1 ml of aqueous solution containing 15% of CuSO4.5H2 O) and then brought to 100° C. for 45 min. The tubes are then cooled in ice before adding 200 μl of Nelson's reagent (aqueous solution of 5.5% of ammonium molybdate, 4.6% of concentrated sulphuric acid and 12% of Na2 SO4.7H2 O).
The volume of the mixture is adjusted to 5 ml with distilled water and its absorbance is measured by spectrophotometry at a wavelength of 500 nm.
The amount of reducing sugars liberated is estimated by comparison with the absorbance values obtained with a calibration series established using a glucose solution. The β-1,3-glucanase enzymatic activity is expressed in nanomoles of glucose equivalent liberated per minute under the conditions of the enzymatic test described above.
2. Isolation and purification of three forms of recombinant β-1,3-glucanase:
a) Method
The different forms of the recombinant protein were isolated and purified from the centrifugate obtained in section 7 (end of the first paragraph). The isolation and purification comprise steps of extraction, ammonium sulphate precipitation, FPLC liquid chromatography on a cation exchange column based on synthetic polymer and exclusion chromatography (molecular sieving) on a crosslinked agarose, according to the protocol described below:
STEP 1:
The bacterial pellet is treated with agitation at 4° C. with a pH 8 buffer solution (30 mM Tris, 1 mM EDTA). After centrifugation (15,000 g, 30 min), the supernatant collected (lysate) constitutes the crude extract.
STEP 2:
The protein extract is precipitated with ammonium sulphate (60% saturation). The proteins which have precipitated are collected by centrifugation (15,000 g for 30 min), solubilised in a buffer solution (100 mM ammonium acetate, pH 5.2) and dialysed overnight at 4° C. against a 100 mM ammonium acetate buffer solution, pH 5.2.
Immediately before proceeding, the concentration of the buffer solution of the protein extract is brought to 10 mM by passage through ready-to-use minicolumns (Pharmacia PD-10).
STEP 3:
The protein extract is then purified by ion exchange chromatography on a column based on synthetic polymer (Pharmacia Mono-S column) according to the Pharmacia FPLC technique.
The extract is applied to the Mono-S column equilibrated with a 10 mM ammonium acetate buffer, pH 5.2. The proteins retained on the column are eluted with a linear gradient from 10 to 500 mM ammonium acetate.
STEP 4:
The fractions containing a β- 1,3-glucanase activity are concentrated by ultrafiltration on a Centricon 10 concentration cartridge (Amicron). The purification of the protein is continued by chromatography (molecular sieving) on a crosslinked agarose (Pharmacia Superose 12 column). Elution is carried out with a 500 mM ammonium acetate buffer, pH 5.2.
At each step, the different forms of recombinant β-1,3-glucanase are identified by their molecular weight (polyacrylamide gel electrophoresis in the presence of SDS--visualisation with silver), by their activities measured by the colorimetric method described above and by their positive reactions with the polyclonal antibodies directed towards tobacco β-1,3-glucanase prepared in section 1.
b) Results
Three forms of the recombinant protein possessing a β-1,3-glucanase activity, of apparent molecular weights 36±3, 37±3 and 39±3 kDa, are thereby isolated and purified. (The molecular weight of the protein deduced from the coding sequence is 37,387 Da). Their respective measured specific activities are 1.65, 1.75 and 1.76 nanomoles of glucose equivalent liberated per minute per microgram of proteins, values which are not significantly different.
3. Determination of the amino-terminal sequence of the three forms of recombinant β-1,3-glucanase.
Sequencing of the amino-terminal ends of the three forms of recombinant β-1,3-glucanase was carried out. The samples to be treated are brought to the surface of a PVDF (polyvinylidene difluoride) filter, which is introduced into a protein sequencer (Model 470 A, marketed by the company Applied Biosystems USA) equipped with a chromatograph (Applied Biosystems Model 430) which continuously analyses the phenylthiohydantoin derivatives formed, after each degradation cycle.
The respective amino-terminal sequences determined are shown below, the symbol X representing an amino acid not determined (SEQ ID NOS:23, 24, and 24, respectively):
______________________________________                                    
Met Ile Gly Val X Tyr Gly Met Leu                                         
Met Ile Gly Val X Tyr Gly Met Leu Gly Asn Asn Leu Pro                     
Met Ile Gly Val X Tyr Gly Met Leu Gly Asn Asn Leu Pro                     
______________________________________
These sequences correspond to the sequence deduced from the coding portion of plasmid pBR59 (see FIGS. 4A-4C). There is hence no post-translational maturation of the amino-terminal portion. The observed differences in molecular weight for a similar specific activity are hence probably the result of a carboxy-terminal post-translational maturation which occurs on 27 amino acids approximately.
Section 9: Construction of a vector for expression in plant cells: the binary vector pBR60.
a) Preparation of a complete recombinant gene for soybean β-1,3-glucanase and cloning thereof into the binary vector pBIN19.
The DNA carrying the coding sequence was obtained by cleavage of the vector M13BR140, obtained in section 6c) (see FIGS. 3A-D and SEQ NO:13), using the restriction enzymes BamHI and SacI, and purified by electrophoresis on low-melting-temperature agarose gel. This DNA was inserted between a promoter sequence comprising so-called 35S promoter of the cauliflower mosaic virus (35S CaMV) and a terminator sequence comprising the nopaline synthase terminator of Agrobacterium tumefaciens.
b) Preparation of the promoter sequence comprising
the 35S promoter of the cauliflower mosaic virus.
The approximately 900-bp HindIII-BamHI fragment containing the 35S promoter is isolated from plasmid pBI121 (Clontech) by cleavage using the endonucleases HindIII and BamHI followed by agarose gel electrophoresis. This fragment is cut again with HindII. The approximately 410-bp fragment carrying the BamHI site is treated with T4 DNA ligase in the presence of a HindIII linker (synthetic sequence containing a HindIII site). After cleavage with the endonuclease HindIII and agarose gel electrophoresis, the resulting HindIII-BamHI fragment (of approximately 420-bp) is isolated and purified.
c) Preparation of the terminator sequence comprising the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens.
An approximately 250-bp fragment containing the nopaline synthase terminator was isolated from plasmid pBI121 (Clontech) by cleavage using the restriction enzymes SacI and EcoRI followed by agarose gel electrophoresis.
The promoter sequence, the coding sequence of the complementary DNA of β-1,3-glucanase and the terminator sequence were ligated using T4 DNA ligase in the binary vector pBIN19 opened using the endonucleases HindIII and EcoRI. This vector carries two genes for resistance to kanamycin, one capable of being expressed in bacteria, and the other, located immediately upstream of the complete recombinant gene (see Bevan, 984, Nucl. Ac. Res., 12, 8711-8721), capable of being transferred to plant cells. The gene for resistance to kanamycin will serve as a selection marker during the steps of transformation and analysis of the progeny of the transformed plants.
The vector obtained is referred to as plasmid pBR60. The nucleotide sequence of the complete recombinant gene, verified by sequencing, together with the peptide sequence deduced are shown in FIGS. 5A-5E. The plasmid is cloned into E. coli strain MC1061 (Clontech).
Section 1: Transfer into Agrobacterium tumefaciens of plasmid pBR60 containing the recombinant gene for β-1,3-glucanase.
a) Transfer into Acrobacterium tumefaciens.
This transfer is carried out as described below by three-way conjugation between the E. coli strain MC1061 containing the vector pBR60 and Agrobacterium tumefaciens strain LBA4404 (Clontech) using E. coli strain HB101 containing the mobilising plasmid pRK2013 (D. M. Figurski et al., 1979, Pro. Ntl. Ac. Sci. USA, 76, 1648-1652).
The E. coli strain MC1061 containing plasmid pBR60 and an E. coli strain HB101 (Clontech) containing the mobilising plasmid pRK2013 are cultured at 37° C. in Luria medium (Gibco) in the presence of 25 mg/l of kanamycin.
Agrobacterium tumefaciens strain LBA4404 is cultured at 28° C. in Luria medium (Gibco) in the presence of 100 mg/l of rifampicin (it is resistant to this antibiotic); 200 μl of each of the three cultures are mixed, plated out on Luria agar medium (Gibco) and incubated overnight at 28° C. The bacteria are then resuspended in 5 ml of Luria medium and aliquot portions are plated out on Petri dishes containing an agar minimum medium (described in 'Plant Molecular Biology Manual" Gelvin et al., Kluwer Academic Press, 1988) in the presence of 100 mg/l of rifampicin and 25 mg/l of kanamycin. Under these conditions, only Agrobacterium tumefaciens colonies which have integrated plasmid pBR60 grow (E. coli strains cannot grow under these conditions). These colonies contain the recombinant gene for β-1,3-glucanase in a context permitting its replication.
The resistance of the selected colonies to both antibiotics is verified by transplanting them on the same selection medium twice in succession. The presence of the recombinant gene for β-1,3-glucanase in Agrobacterium tumefaciens is verified by the Southern blot method on a total DNA preparation (cell lysis, purification of the DNA by extraction using a phenol/chloroform mixture, according to the protocol described by Gelvin in the work cited above, cleavage of the purified DNA using restriction enzymes, agarose gel electrophoresis, transfer onto a membrane and hybridisation according to techniques well-known to those skilled in the art).
b) Transfer into Agrobacterium rhizogenes
This transfer is carried out in the same manner as the transfer into Agrobacterium tumefaciens described in a), with Agrobacterium rhizogenes strain A4 described by GUERCHE et al., (1987) Mol. gen. genet. 206, 382.
Section 11: Obtaining transformed tobacco plants
Nicotiana tabacum tobacco cultured in vitro was infected with Agrobacterium tumefaciens containing plasmid pBR60 according to the procedure of Horsch et al., well-known to a person skilled in the art (Horsch R. B. et al., 1985, Science 227, 1229-1231), the main steps of which are described below.
Discs of axenic N. tabacum tobacco plants (variety Wisconsin Havana 38, sensitive to pathogenic fungi) are incubated in a culture of A. tumefaciens harbouring plasmid pBR60. The discs, drained on Whatman paper, were transferred onto culture media in Petri dishes in order to multiply the transformed cells so as to obtain calluses, and then to produce buds in the presence of cefotaxime (500 μg/ml), which is designed to remove Agrobacterium tumefaciens, and kanamycin (100 μg/ml).
Buds resistant to kanamycin were then transferred onto a medium permitting the induction of roots in the presence of cefotaxime and kanamycin. The plantlets are then transplanted into pots in a substrate composed of peat and compost, and left to grow in a greenhouse. All the transformed plants (R0 generation) which survived the steps of regeneration and acclimatisation in the greenhouse proved morphologically normal and fertile. They were self-fertilised and gave seeds (R1 generation).
Section 12; Analysis of the genomic DNA of the transformed tobacco plants (R0 generation) according to the Southern blot technique.
The high molecular weight genomic DNA was isolated from mature leaves of transgenic plants of the R0 generation according to the method of extraction using cetyltrimethylammonium bromide and of purification by precipitation, described in the work "Plant Molecular Biology Manual" already cited.
10 μg of this genomic DNA were digested overnight at 37° C. with 20 units of the restriction enzymes HindIII and EcoRI. The restriction fragments obtained were separated by electrophoresis on agarose gel (1%). The DNA was transferred according to the Southern blot method onto a Nylon filter (Amersham Hybond N+), and hybridised with a nucleotide probe comprising a portion of the sequence of the recombinant gene, labelled by coupling to peroxidase (ECL kit, Amersham). The membranes are then washed and visualised according to the protocol recommended by Amersham.
Analysis of the films enables the following conclusions to be drawn:
some plants do not possess copies of the transferred recombinant gene (absence of signal),
most of the plants tested contain at least one copy without rearrangement of the construction: 35S CaMV promoter--coding sequence of the complementary DNA of β-1,3-glucanase--NOS terminator,
some profiles suggest that there are internal rearrangements in the construction, but these events are rare.
Section 13: Demonstration of the expression of soybean β-1,3-glucanase in transformed tobacco plants (of RO generation).
a) Preparation of crude protein extracts of transformed tobacco plants (R0 generation).
Crude protein extracts were prepared from various plant tissues (root, stem, leaf, and the like). The tissue fragments were frozen in liquid nitrogen, reduced to powder and stored at -20° C. The powder was extracted at 4° C. in the presence of a 0.1M ammonium acetate buffer, pH 5.2, and subjected to a centrifugation at 10,000 g. The total protein concentration was determined on the supernatants, hereinafter referred to as crude protein extracts, following the technique of Bradford (Bradford, M. M., 1976, Anal. Biochem., 72, 248-254).
b) Demonstration by immunoblotting (Western blot)
The crude protein extracts of different transformed plants and of untransformed (control) plants are subjected to a Western blot, a technique well-known to a person skilled in the art and described, in particular, by H. Towbin et al., Proc. Ntl. Acad. Sci. USA, 76, 1972, 4350-4354, which comprises the following steps:
denaturation by heating to 100° C. for 10 min in a buffer, called loading buffer, consisting of 0.125M Tris-HCl, pH 6.8, 4% SDS, 0,002% bromophenol blue, 20% glycerol, 10% β-mercaptoethanol (according to the protocol described by Laemmli, U. K. Laemmli, Nature, 227, 1970, 680-685);
electrophoretic separation of the different proteins contained in the solubilisate according to the protocol described by Laemmli (ref. above);
electrotransfer of the said proteins contained in the gel onto a PVDF membrane (according to the technique of H. Towbin et al., Proc. Natl. Acad. Sci. USA 76, 1979, 4350-4354).
Immunodetection is carried out according to a protocol which comprises the following steps:
saturation of the PVDF membrane onto which the proteins have been transferred by incubation for not less than 2 hours at 37° C. in a 3% solution of gelatin,
3 washes in phosphate-buffered saline containing 0.05% of Tween 20 detergent,
incubation (for 1 hour at 37° C.) in the presence of the immune serum prepared above (containing polyclonal antibodies which recognise the recombinant protein) diluted to 1/10,000 in phosphate-buffered saline,
3 washes in phosphate-buffered saline containing 0.05% of Tween 20 detergent.
The antigen-antibody complex is then visualised using an alkaline phosphatase-conjugated streptavidin/biotin system with the Amersham kit RPN 23 ("Blotting detection kit"), used according to the manufacturer's directions.
The blot obtained shows the presence of a protein of approximately 37±3 kDa for the transformed plants which is absent from the control plants (the protein deduced from cDNA sequence, from which its assumed signal peptide for an expression in eukaryotic cells has been cleaved, has a molecular weight of 38,156 Da).
Analysis according to the Western blot technique was performed on 30 transformed plants (responding positively to Southern blot). 28 plants showed an expression of the recombinant β-1,3-glucanase in Western blot.
c) Comparison of the electrophoretic migrations of soybean β-1,3-glucanases (recombinant proteins and natural protein).
The apparent molecular weight obtained for the recombinant protein expressed in the transformed tobacco plants was compared with that obtained for the natural protein present in an extract of soybean cells (see section 1) and natural tobacco β-1,3-glucanase present in an extract of tobacco cells (see section 1), by electrophoretic migration on parallel lanes according to the protocol described by Laemmli (U. K. Laemmli, Nature 227, 1970, 680-685). The sizes of these proteins were compared with those of proteins of known molecular masses (molecular weight markers of between 14,000 and 97,400 Da of Bio-Rad--ref. 61-0304).
The blots obtained after Western blot show that soybean β-1,3-glucanase synthesised in tobacco possesses the same apparent molecular weight as that of the natural soybean protein: 37±3 kDa. The post-translational maturation of this protein is hence performed in the same manner in a plant other than soybean. This apparent molecular weight of soybean β-1,3-glucanase is approximately 3 kDa higher than that of tobacco β-1,3-glucanase, whose molecular weight is 34,969 Da (H. Shinshi et al., 1988, Proc. Natl. Aca. Sci. USA, 85, 5541-5545). The molecular weight of soybean β-1,3-glucanase is hence in the region of 38 kDa, which is very close to the molecular weight predicted in section 5 for the mature protein of 338 amino acids predicted in section 5, which is 38,156 Da.
Section.14: Measurement of the resistance of transformed formed tobacco plants (R1 generation) to pathogenic fungi.
Tobacco plants regenerated (R0 generation) in the presence of kanamycin were self-pollinated. The mature seeds (R1 generation) are sown on Murashige and Skoog medium supplemented with 100 μg/ml of kanamycin.
The kanamycin-resistant plantlets of the R1 generation, derived from 13 chosen transformed plants, from a Nicotiana tabacum var. Wisconsin Havana 38 plant sensitive to the fungus Chalara elegans (also known as Thielaviopsis basicola), abbreviated to W38, were transferred to the greenhouse for evaluation of their resistance to this fungus. The latter was chosen since it is representative of the pathogenic fungi of tobacco possessing β-1,3-glucans in their wall. The study covered numbers of plantlets varying from 16 to 25 depending on the progeny of the transformed plants. The protocol chosen in this study is described below:
The plantlets are cultivated in pots (3×3 cm). On appearance of the 5th leaf, the plants are inoculated by depositing a suspension of 5×105 endoconidia of this fungus on the neck.
The endoconidia are taken from mycelial cultures of this fungus maintained on potato dextrose agar medium (Difco) at 22° C. and in the dark. The resistance to Chalara elegans is evaluated 45 days after inoculation.
The plants are scored according to the symptoms of infection and according to their level of vegetative development.
Two scorings are implemented:
Measurement of the weight (in grams) of the aerial parts of the plants
Measurement of a resistant index taking into account the impact of the disease on the whole of the plant.
The classes are defined according to the following criteria:
Score 0: plant dead
Score 1: terminal bud still green, root system destroyed
Score 2: development of the plants not exceeding 25% of that of the control, root system completely necrotic
Score 3: development of the plants attaining 50% of the development of the control, root system displaying healthy portions
Score 4: development of the plants identical to the control.
The resistance index of the progeny of a transformed plant represents the mean of the scores assigned to the plantlets derived from this plant.
The table below collates the main results obtained:
              TABLE 1                                                     
______________________________________                                    
Measurement of the resistance of progeny of                               
transformed tobacco plants to pathogenic fungi                            
         Number  Resistance Weight of the                                 
         tested  Index      aerial parts                                  
______________________________________                                    
Transformed                                                               
plants                                                                    
No. 5      24        1          0.22                                      
No. 10     23        0.87       0.22                                      
No. 17     21        1.04       0.29                                      
No. 20     17        1.41       0.50                                      
No. 22     23        1          0.35                                      
No. 29     19        1.1        0.46                                      
No. 30     16        0.5        0.14                                      
No. 37     20        0.25       0.46                                      
No. 43     25        0.56       0.075                                     
No. 45     23        0.30       0.65                                      
No. 49     21        0.19       0.72                                      
No. 55     22        0.09       0.05                                      
No. 59     25        0.48       0.14                                      
Control plants                                                            
W38        25        0.12       0.06                                      
______________________________________                                    
 W38: Untransformed Nicotiana tabacum var. Wisconsin Havana 38 plant
It is seen on studying the above table that 12/13 of the progeny of transformed tobacco plants have a resistance index and a weight of the aerial parts which are higher than those of the progeny of the untransformed W38 plant.
The transformation of tobacco plants with the recombinant DNA of the invention hence confers on their progeny an increased resistance to pathogenic fungi.
Section 15: Purification of the recombinant β-1,3-glucanase from the leaves of transformed tobacco plants (R1 generation) and determination of its amino-terminal sequence.
1) Purification of the recombinant β-1,3-glucanase
The recombinant protein was purified from crude extracts of transformed tobacco leaf proteins, by ammonium sulphate precipitation and FPLC liquid chromatography on a cation exchange column based on synthetic polymer on a crosslinked agarose, according to the protocol described below:
Protocol for purification of the recombinant β-1,3-glucanase
STEP 1: The protein extract is precipitated with ammonium sulphate (60% saturation). The proteins which have precipitated are recovered by centrifugation (15,000 g for 30 min), solubilised in a buffer solution (100 mM ammonium acetate, pH 5.2) and dialysed overnight at 4° C. against a 100 mM ammonium acetate buffer solution, pH 5.2.
Immediately before proceeding, the concentration of the buffer solution of the protein extract is brought to 10 mM by passage through ready-to-use minicolumns (Pharmacia PD-10).
STEP 2: The protein extract is then purified by ion exchange chromotography based on synthetic polymer (Pharmacia Mono-S column) using an FPLC technique (Pharmacia).
The extract is applied to the Mono-S column equilibrated with a 10 mM ammonium acetate buffer, pH 5.2. The proteins retained on the column are eluted with a linear gradient from 10 to 500 mM ammonium acetate.
At each step, the soybean β-1,3-glucanase is identified by its molecular weight (polyacrylamide gel electrophoresis in the presence of SDS--visualisation with silver), and by its immunoblot (see Section 13b)) and its activity, measured by the colorimetric method described in Section 8 1).
2) Determination of the amino-terminal sequence of the recombinant β-1,3-glucanase
After purification of the recombinant β-1,3-glucanase according to the protocol described above, sequencing of the amino-terminal end was carried out. The samples to be treated are brought to the surface of a PVDF (polyvinylidene difluoride) filter by electrotransfer according to the method described by H. TOWBIN et al. Proc. Ntl. Acad. Sci. USA (1979), 4350-4354, after polyacrylamide gel electrophoresis in the presence of SDS. The filter is introduced into a protein sequencer (model 470 A marketed by the company Applied Biosystems (USA)) equiped with a chromatograph (Applied Biosystems model 430) which continuously analyses the phenylthiohydantion derivatives formed, after each degradation cycle.
It was not possible to determine an amino-terminal sequence, despite the good operation of the sequencer, checked by the determination of the amino-terminal sequence of a control protein: the lactoglobulin.
It is thus probable that the amino-terminal sequence of the recombinant β-1,3-glucanase begins with a Gln, as predicted by comparison of the already determined amino-terminal sequences of dicotyledons (see section 5).
It has indeed already been shown that the amino-terminal Gln was blocked for another soybean β-1,3-glucanase (TAKEUCHI et al., 1990, Plant Physiol. 93, 673-682).
Section 16: Obtaining transformed rape plants.
The transformation is carried out according to the protocol of P. Guerche et al. (P. Guerche et al., 1987, Mol. Gen. Genet., 206, 382). The different culture media are those described by Pelletier et al. (Pelletier et al., 1983, Mol. Gen. Genet., 191, 244). Their composition is detailed below (Table 2).
a) Obtaining transformed roots.
Stem segments are removed from the apical tip of rape plants (Brassica napus: spring varieties Brutor and Westar and winter variety) approximately 1 m in height. These segments are surface sterilised, rinsed in sterile water, cut into segments 1.5 cm in length approximately and placed in a tube containing medium A.
Inoculation of the tip of this segment is performed by application of a suspension of the Agrobacterium rhizogenes strain containing plasmid pBR60.
Transformed roots appear on the stem segment after 1 to 2 weeks; they are removed and placed on agar medium B (15 g/l) supplemented with 500 μg of cefotaxime/ml.
b) Regeneration of transformed plants.
Root fragments are incubated for 15 days on medium D containing 3 mg/l of 2,4-dichlorophenoxyacetic acid, and then placed on RCC medium for induction of buds. Rooted plants are then obtained by passage of the buds through media F and G.
Section 17: Demonstration of the expression of soybean β-1,3-glucanase in transformed rape plants.
a) Preparation of crude protein extracts of transformed rape plants (R0 generation)
Crude protein extracts were prepared from leaves of the plant. These extracts were frozen in liquid nitrogen, reduced to powder and stored at -20° C. The powder was extracted at 4° C. in the presence of a 0.4M ammonium acetate buffer, pH 5.2, and subjected to a centrifugation at 10,000 g. The total protein concentration was determined on the supernatants, hereinafter referred to as crude protein extracts, following the technique of Bradford (Bradford, M. M., 1976, Anal. Biochem., 72,248-254).
b) Demonstration by immunoblotting (Western blot)
The crude protein extracts of different transformed plants and of untransformed (control) plants are subjected to a Western blot, a technique well-known to a person skilled in the art and described above.
The antigen-antibody complex is then visualised using an alkaline phosphatase-conjugated streptavidin/biotin system with Amersham kit RPN 23 ("Blotting detection kit"), used according to the manufacturer's directions.
The blot obtained shows the presence of a protein of approximately 37±3 kDa for the transformed plants, which is absent from the control plants.
Analysis according to the Western blot technique was performed on 30 transformed plants (responding positively to Southern blot). 38 plants showed an expression of the recombinant β-1,3-glucanase in Western blot.
                                  TABLE 2                                 
__________________________________________________________________________
Composition of the different media used for obtaining transformed rape    
plants                                                                    
           Medium                                                         
Composition                                                               
(mg/1)     A    B    RCC  F    G                                          
__________________________________________________________________________
NH.sub.4 NO.sub.3                                                         
           1,650     1,650                                                
                          1,650                                           
                               825                                        
KNO.sub.3  1,900                                                          
                2,500                                                     
                     1,900                                                
                          1,900                                           
                               950                                        
(NH.sub.4).sub.2 SO.sub.4                                                 
                134                                                       
NaH.sub.2 PO.sub.4                                                        
                150                                                       
KH.sub.2 PO.sub.4                                                         
           170       170  170  85                                         
CaCl.sub.2.2H.sub.2 O                                                     
           440  750  440  440  220                                        
MgSO.sub.4.7H.sub.2 O                                                     
           370  250  370  370  185                                        
H.sub.3 BO.sub.3                                                          
           12.4 3    12.4 6.2  6.2                                        
MnSO.sub.4.4H.sub.2 O                                                     
           33.6 10   33.6 22.3 22.3                                       
ZnSO.sub.4.7H.sub.2 O                                                     
           21   2    21   8.6  8.6                                        
KI         1.66 0.75 1.66 0.83 0.83                                       
Na.sub.2 MoO.sub.4.2H.sub.2 O                                             
           0.5  0.25 0.5  0.25 0.25                                       
CuSo.sub.4.5H.sub.2 O                                                     
           0.05 0.025                                                     
                     0.05 0.25 0.25                                       
CoCl.sub.2.6H.sub.2 O                                                     
           0.05 0.025                                                     
                     0.05 0.25 0.25                                       
FeSO.sub.4.7H.sub.2 O                                                     
           22.24                                                          
                27.8 27.8 27.8 22.24                                      
Na.sub.2 EDTA                                                             
           29.84                                                          
                37.3 37.3 37.3 29.84                                      
Inositol   100  100  100  100  100                                        
Nicotinic acid                                                            
           0.5  1    0.5  1    0.5                                        
Pyridoxine HCl                                                            
           0.5  1    0.5  1    0.5                                        
Thiamine        10        10                                              
Glycine    2         2         2                                          
Glucose    10,000                                                         
                20,000         10,000                                     
Sucrose    10,000    10,000                                               
                          10,000                                          
D-mannitol      70,000                                                    
                     10,000                                               
NAA             1    1    0.1  0.1                                        
BA              1    0.5  0.5                                             
2,4D            0.25                                                      
Adenine sulphate                                                          
IPA                  0.5                                                  
GA.sub.3             0.02                                                 
Tween 80        10                                                        
Agar       8,000     8,000                                                
                          8,000                                           
                               8,000                                      
pH         5.8  5.8  5.8  5.8  5.8                                        
Gentamicin (sulphate)                                                     
           10                                                             
__________________________________________________________________________
 NAA = naphthaleneacetic acid                                             
 BA = 6benzylaminopurine                                                  
 2,4D = 2,4dichlorophenoxyacetic acid                                     
 IPA = N.sup.6(2-isopentenyl)adenine                                      
 GA.sub.3 = gibberellic acid                                              
 EDTA = ethylenediaminetetraacetic acid                                   

__________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 26                                             
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 309 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
IleGlyValCys TyrGlyMetLeuGlyAsnAsnLeuProSerAlaAsn                         
151015                                                                    
AspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArgLeu                          
20 2530                                                                   
TyrAspProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSerGlyIle                          
354045                                                                    
GluLeuIleLeuGlyValProAsnSerAspLeu GlnGlyLeuAlaThr                         
505560                                                                    
AsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsnPhe                          
6570758 0                                                                 
TrpProSerValLysIleLysTyrValAlaValGlyAsnGluValSer                          
859095                                                                    
ProValGlyGlySerSerSerValAlaGlnTyrValLeuProAlaIle                          
 100105110                                                                
GlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAspGlnIle                          
115120125                                                                 
LysValSerThrSerI leAspMetThrLeuIleGlyAsnSerPhePro                         
130135140                                                                 
ProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAspPro                          
145150 155160                                                             
IleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeuValAsnVal                          
165170175                                                                 
TyrProTyrPheSerTyrThrGlyAsnProAr gAspIleSerLeuPro                         
180185190                                                                 
TyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGlnTyr                          
195200205                                                                 
GlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHisAlaAla                          
210215220                                                                 
IleAspAsnThrLysIleGlyTyrValGluValValValSerGluSer                          
225 230235240                                                             
GlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsnAlaArg                          
245250255                                                                 
ValTyrLeuAspA snLeuValArgArgAlaAsnArgGlySerProArg                         
260265270                                                                 
ArgProSerLysProThrGluThrTyrIlePheAlaMetPheAspGlu                          
275 280285                                                                
AsnGlnLysAsnProGluIleGluLysHisPheGlyLeuPheAsnPro                          
290295300                                                                 
AsnLysGlnLysLys                                                           
305                                                                       
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
 (A) LENGTH: 28 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
TyrProPheGlyPheGlyGlyLysArgLeuGlyLysValValIleAsp                          
1510 15                                                                   
AspPheAsnAlaThrThrSerIleLysSerAspVal                                      
2025                                                                      
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 338 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
GlnIleGlyValCysTyrGlyMetLeuGlyAsnAsnLeuProSerAla                          
151015                                                                    
AsnAspValIleGlyLeuTyrArgSerAsnAsnIleL ysArgMetArg                         
202530                                                                    
LeuTyrAspProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSerGly                          
354045                                                                    
IleG luLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAla                         
505560                                                                    
ThrAsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsn                          
6570 7580                                                                 
PheTrpProSerValLysIleLysTyrValAlaValGlyAsnGluVal                          
859095                                                                    
SerProValGlyGlySerSerS erValAlaGlnTyrValLeuProAla                         
100105110                                                                 
IleGlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAspGln                          
115120 125                                                                
IleLysValSerThrSerIleAspMetThrLeuIleGlyAsnSerPhe                          
130135140                                                                 
ProProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAsp                          
 145150155160                                                             
ProIleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeuValAsn                          
165170175                                                                 
Val TyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeu                         
180185190                                                                 
ProTyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGln                          
195 200205                                                                
TyrGlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHisAla                          
210215220                                                                 
AlaIleAspAsnThrLysIleGlyTyrValG luValValValSerGlu                         
225230235240                                                              
SerGlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsnAla                          
245250 255                                                                
ArgValTyrLeuAspAsnLeuValArgArgAlaAsnArgGlySerPro                          
260265270                                                                 
ArgArgProSerLysProThrGluThrTyrIlePheAlaMe tPheAsp                         
275280285                                                                 
GluAsnGlnLysAsnProGluIleGluLysHisPheGlyLeuPheAsn                          
290295300                                                                 
ProAsnLysGln LysLysTyrProPheGlyPheGlyGlyLysArgLeu                         
305310315320                                                              
GlyLysValValIleAspAspPheAsnAlaThrThrSerIleLysSer                          
3 25330335                                                                
AspVal                                                                    
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 25 amino acids                                                
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                   
MetAlaProSerGlyLysSer ThrLeuLeuLeuLeuPheLeuLeuLeu                         
151015                                                                    
CysLeuProSerTrpAsnAlaGlyAla                                               
2025                                                                      
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 89 amino acids                                                
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                   
MetSerSerProLeuLysAsnAlaLeuValThrAlaMetLeuAlaGly                          
1510 15                                                                   
GlyAlaLeuSerSerProThrLysGlnHisValGlyIleProValAsn                          
202530                                                                    
AlaSerProGluValGlyProGlyLysTyrSerPheLysGln ValArg                         
354045                                                                    
AsnProAsnTyrLysPheAsnGlyProLeuSerValLysLysThrTyr                          
505560                                                                    
LeuLysTyrGlyVal ProIleProAlaTrpLeuGluAspAlaValGln                         
65707580                                                                  
AsnSerThrSerGlyLeuAlaGluArg                                               
85                                                                        
(2) INFORMATION FOR SEQ ID NO:6:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
 (A) LENGTH: 24 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                   
MetArgArgThrSerLysLeuThrThrPheSerLeuLeuPheSerLeu                          
15 1015                                                                   
ValLeuLeuSerAlaAlaLeuAla                                                  
20                                                                        
(2) INFORMATION FOR SEQ ID NO:7:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 32 amino acids                                                
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                   
MetProSerLeuPheAlaArgAsnGlnArgPheSerLeuAlaThrLeu                          
151015                                                                    
LeuLeuLeuLeuGluLeuLeuThrGlyAsnLeuArgMet AlaAspAla                         
202530                                                                    
(2) INFORMATION FOR SEQ ID NO:8:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 927 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
( xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                  
ATTGGTGTGTGTTATGGCATGCTGGGCAACAATCTACCGTCAGCAAACGATGTTATAGGT60            
CTTTATAGATCAAATAACATAAAGAGAATGAGACTCTATGATCCTAATCAAGCTGCTCTA120           
GAAGCACTTAGAAATTCTGGCATTGAACTCATT CTTGGGGTGCCAAACTCTGACCTTCAA180          
GGCCTTGCCACCAATCCTGACACTTCTCGTCAATGGGTGCAAAAAAACGTGTTGAACTTT240           
TGGCCTAGTGTCAAAATCAAGTACGTGGCAGTTGGAAATGAAGTGAGTCCCGTTGGAGGC300           
TCTTCTTC GGTAGCCCAATATGTTCTACCTGCCATCCAAAATGTATACCAAGCAATAAGA360          
GCTCAAGGCCTTCATGATCAAATCAAGGTTTCAACATCTATTGACATGACCCTAATAGGA420           
AACTCTTTCCCTCCATCGCAAGGTTCCTTCAGGGGTGATGTGAGATCATAC CTAGATCCC480          
ATAATTGGGTACTTGGTATATGCAAATGCACCATTACTAGTCAATGTGTACCCTTATTTT540           
AGTTACACTGGTAACCCCCGTGACATATCACTTCCCTATGCTCTTTTCACAGCACCAAAT600           
GTTGTGGTATGGGATGGTCAATATGG GTACCAAAATTTGTTTGATGCTATGTTGGATTCA660          
GTACATGCAGCCATTGATAACACTAAGATTGGTTATGTGGAGGTTGTTGTATCCGAGAGT720           
GGGTGGCCATCAGATGGAGGATTTGCTGCCACTTATGACAACGCACGCGTGTACTTAGAC780           
 AATTTGGTTCGTCGTGCTAATAGAGGAAGCCCAAGAAGGCCTTCGAAGCCCACTGAGACT840          
TATATATTTGCCATGTTCGATGAAAATCAAAAAAATCCAGAGATAGAGAAACATTTTGGG900           
CTCTTCAATCCCAACAAACAAAAAAAA 927                                           
(2) INFORMATION FOR SEQ ID NO:9:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 96 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                   
ATGCCTTCTCTCTTCGCTAGAAACCAGAGGTTCT CATTGGCTACTCTCCTGCTTCTTCTG60           
GAACTATTGACAGGAAACCTTCGCATGGCAGATGCT96                                    
(2) INFORMATION FOR SEQ ID NO:10:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 84 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
 (D) TOPOLOGY: linear                                                     
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                  
TACCCATTTGGGTTTGGAGGAAAGAGGCTAGGGAAAGTTGTTATTGACGACTTCAATGCA60            
ACAACTTCCATTAAGAGTGATGTG84                                                
(2) INFORMATION FOR SEQ ID NO:11:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1483 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(iii) HYPOTHETICAL: NO                                                    
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 114..1223                                                   
(ix) FEATURE:                                                             
 (A) NAME/KEY: mat.sub.-- peptide                                         
(B) LOCATION: 213..1223                                                   
(ix) FEATURE:                                                             
(A) NAME/KEY: sig.sub.-- peptide                                          
(B) LOCATION: 114..212                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                  
AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTGGCGATCATCAAGCCTAATA60            
GAGGGCTAATCCTTCACTTG TTTGTTTTGTGGTGTATTATTACATTTTGCACCATG116              
Met                                                                       
33                                                                        
CCTTCTCTCTTC GCTAGAAACCAGAGGTTCTCATTGGCTACTCTCCTG164                      
ProSerLeuPheAlaArgAsnGlnArgPheSerLeuAlaThrLeuLeu                          
30-25-20                                                                  
CTTCTTCTGGAACTA TTGACAGGAAACCTTCGCATGGCAGATGCTCAA212                      
LeuLeuLeuGluLeuLeuThrGlyAsnLeuArgMetAlaAspAlaGln                          
15-10-5                                                                   
ATTGGTGTGTGTTATGGCATG CTGGGCAACAATCTACCGTCAGCAAAC260                      
IleGlyValCysTyrGlyMetLeuGlyAsnAsnLeuProSerAlaAsn                          
151015                                                                    
GATGTTATAGGTCTTTAT AGATCAAATAACATAAAGAGAATGAGACTC308                      
AspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArgLeu                          
202530                                                                    
TATGATCCTAATCAAGCT GCTCTAGAAGCACTTAGAAATTCTGGCATT356                      
TyrAspProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSerGlyIle                          
354045                                                                    
GAACTCATTCTTGGGGTGCCA AACTCTGACCTTCAAGGCCTTGCCACC404                      
GluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAlaThr                          
505560                                                                    
AATCCTGACACTTCTCGTCAATGGGTG CAAAAAAACGTGTTGAACTTT452                      
AsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsnPhe                          
65707580                                                                  
TGGCCTAGTGTCAAAATCAAG TACGTGGCAGTTGGAAATGAAGTGAGT500                      
TrpProSerValLysIleLysTyrValAlaValGlyAsnGluValSer                          
859095                                                                    
CCCGTTGGAGGCTCTTCT TCGGTAGCCCAATATGTTCTACCTGCCATC548                      
ProValGlyGlySerSerSerValAlaGlnTyrValLeuProAlaIle                          
100105110                                                                 
CAAAATGTATACCAAGCA ATAAGAGCTCAAGGCCTTCATGATCAAATC596                      
GlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAspGlnIle                          
115120125                                                                 
AAGGTTTCAACATCTATTGAC ATGACCCTAATAGGAAACTCTTTCCCT644                      
LysValSerThrSerIleAspMetThrLeuIleGlyAsnSerPhePro                          
130135140                                                                 
CCATCGCAAGGTTCCTTCAGGGGTGAT GTGAGATCATACCTAGATCCC692                      
ProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAspPro                          
145150155160                                                              
ATAATTGGGTACTTGGTATAT GCAAATGCACCATTACTAGTCAATGTG740                      
IleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeuValAsnVal                          
165170175                                                                 
TACCCTTATTTTAGTTAC ACTGGTAACCCCCGTGACATATCACTTCCC788                      
TyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeuPro                          
180185190                                                                 
TATGCTCTTTTCACAGCA CCAAATGTTGTGGTATGGGATGGTCAATAT836                      
TyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGlnTyr                          
195200205                                                                 
GGGTACCAAAATTTGTTTGAT GCTATGTTGGATTCAGTACATGCAGCC884                      
GlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHisAlaAla                          
210215220                                                                 
ATTGATAACACTAAGATTGGTTATGTG GAGGTTGTTGTATCCGAGAGT932                      
IleAspAsnThrLysIleGlyTyrValGluValValValSerGluSer                          
225230235240                                                              
GGGTGGCCATCAGATGGAGGA TTTGCTGCCACTTATGACAACGCACGC980                      
GlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsnAlaArg                          
245250255                                                                 
GTGTACTTAGACAATTTG GTTCGTCGTGCTAATAGAGGAAGCCCAAGA1028                     
ValTyrLeuAspAsnLeuValArgArgAlaAsnArgGlySerProArg                          
260265270                                                                 
AGGCCTTCGAAGCCCACT GAGACTTATATATTTGCCATGTTCGATGAA1076                     
ArgProSerLysProThrGluThrTyrIlePheAlaMetPheAspGlu                          
275280285                                                                 
AATCAAAAAAATCCAGAGATA GAGAAACATTTTGGGCTCTTCAATCCC1124                     
AsnGlnLysAsnProGluIleGluLysHisPheGlyLeuPheAsnPro                          
290295300                                                                 
AACAAACAAAAAAAATACCCATTTGGG TTTGGAGGAAAGAGGCTAGGG1172                     
AsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArgLeuGly                          
305310315320                                                              
AAAGTTGTTATTGACGACTTC AATGCAACAACTTCCATTAAGAGTGAT1220                     
LysValValIleAspAspPheAsnAlaThrThrSerIleLysSerAsp                          
325330335                                                                 
GTGTAAGGTTGGAATCCTACT CCTCAAAATCTCTGTTATTCCACCCATAAAAT1273                
Val                                                                       
AAGAGAGAATATGTTGTTTGTGTGAAATATGTATATATCCTTCAGTCTTGGATGAATAAA1333          
ATTTGTGAAAATTTTATTTTTTTTTTTTTTGGACTAGAAATAGCCTGATACTTAATTATT 1393         
ATCTTTTTATACCACACGTTGGTTTCCTTCATGAGTACAAACCGAAATAAAACCAACAAT1453          
TAATCTTGTTTTATTACAACACACAAGCTT1483                                        
(2) INFORMATION FOR SEQ ID NO:12:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
( A) LENGTH: 370 amino acids                                              
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                  
MetProSerLeuPheAlaArgAsnGlnArgPheSerLeuAlaThrLeu                          
33-30-25- 2 0                                                             
LeuLeuLeuLeuGluLeuLeuThrGlyAsnLeuArgMetAlaAspAla                          
15-10-5                                                                   
GlnIleGlyValCysTyrGlyMetLeuGlyAsnAsnLeuProSerAla                          
1 51015                                                                   
AsnAspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArg                          
202530                                                                    
LeuTyrAsp ProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSerGly                         
354045                                                                    
IleGluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAla                          
50 5560                                                                   
ThrAsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsn                          
657075                                                                    
PheTrpProSerValLysIleLysTyrValAlaValG lyAsnGluVal                         
80859095                                                                  
SerProValGlyGlySerSerSerValAlaGlnTyrValLeuProAla                          
100105 110                                                                
IleGlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAspGln                          
115120125                                                                 
IleLysValSerThrSerIleAspMetThrLeuIleGlyAsnSerPh e                         
130135140                                                                 
ProProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAsp                          
145150155                                                                 
ProIleIleGlyTyrLeu ValTyrAlaAsnAlaProLeuLeuValAsn                         
160165170175                                                              
ValTyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeu                          
180 185190                                                                
ProTyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGln                          
195200205                                                                 
TyrGlyTyrGlnAsnLeuPheAspAlaM etLeuAspSerValHisAla                         
210215220                                                                 
AlaIleAspAsnThrLysIleGlyTyrValGluValValValSerGlu                          
225230235                                                                 
 SerGlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsnAla                         
240245250255                                                              
ArgValTyrLeuAspAsnLeuValArgArgAlaAsnArgGlySerPro                          
 260265270                                                                
ArgArgProSerLysProThrGluThrTyrIlePheAlaMetPheAsp                          
275280285                                                                 
GluAsnGln LysAsnProGluIleGluLysHisPheGlyLeuPheAsn                         
290295300                                                                 
ProAsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArgLeu                          
3053 10315                                                                
GlyLysValValIleAspAspPheAsnAlaThrThrSerIleLysSer                          
320325330335                                                              
AspVal                                                                    
(2) INFORMATION FOR SEQ ID NO:13:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
 (A) LENGTH: 1483 base pairs                                              
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(iii) HYPOTHETICAL: NO                                                    
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 114..1223                                                   
(ix) FEATURE:                                                             
(A) NAME/KEY: sig.sub.-- peptide                                          
(B) LOCATION: 114..212                                                    
(ix) FEATURE:                                                             
(A) NAME/KEY: mat.sub.-- peptide                                          
(B) LOCATION: 213..1223                                                   
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                  
AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTGGCGATCATCAAGCCTAATA60            
GAGGGCTAATCCTTCACTTGTTTGTTTTGTGGTGTATTCATATGTTTGG ATCCATG116              
Met                                                                       
33                                                                        
CCTTCTCTCTTCGCTAGAAACCAGAGGTTCTCATTG GCTACTCTCCTG164                      
ProSerLeuPheAlaArgAsnGlnArgPheSerLeuAlaThrLeuLeu                          
30-25-20                                                                  
CTTCTTCTGGAACTATTGACAGGAAACCTTCGCATGGCA GATGCTCAA212                      
LeuLeuLeuGluLeuLeuThrGlyAsnLeuArgMetAlaAspAlaGln                          
15-10-5                                                                   
ATTGGTGTGTGTTATGGCATGCTGGGCAACAATCTACCGTCAGCA AAC260                      
IleGlyValCysTyrGlyMetLeuGlyAsnAsnLeuProSerAlaAsn                          
151015                                                                    
GATGTTATAGGTCTTTATAGATCAAATAACATAAAGAGAATG AGACTC308                      
AspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArgLeu                          
202530                                                                    
TATGATCCTAATCAAGCTGCTCTAGAAGCACTTAGAAATTCT GGCATT356                      
TyrAspProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSerGlyIle                          
354045                                                                    
GAACTCATTCTTGGGGTGCCAAACTCTGACCTTCAAGGCCTTGCC ACC404                      
GluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAlaThr                          
505560                                                                    
AATCCTGACACTTCTCGTCAATGGGTGCAAAAAAACGTGTTGAACTTT 452                      
AsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsnPhe                          
65707580                                                                  
TGGCCTAGTGTCAAAATCAAGTACGTGGCAGTTGGAAATGAAGTG AGT500                      
TrpProSerValLysIleLysTyrValAlaValGlyAsnGluValSer                          
859095                                                                    
CCCGTTGGAGGCTCTTCTTCGGTAGCCCAATATGTTCTACCT GCCATC548                      
ProValGlyGlySerSerSerValAlaGlnTyrValLeuProAlaIle                          
100105110                                                                 
CAAAATGTATACCAAGCAATAAGAGCACAAGGCCTTCATGAT CAAATC596                      
GlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAspGlnIle                          
115120125                                                                 
AAGGTTTCAACATCTATTGACATGACCCTAATAGGAAACTCTTTC CCT644                      
LysValSerThrSerIleAspMetThrLeuIleGlyAsnSerPhePro                          
130135140                                                                 
CCATCGCAAGGTTCCTTCAGGGGTGATGTGAGATCATACCTAGATCCC 692                      
ProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAspPro                          
145150155160                                                              
ATAATTGGGTACTTGGTATATGCAAATGCACCATTACTAGTCAAT GTG740                      
IleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeuValAsnVal                          
165170175                                                                 
TACCCTTATTTTAGTTACACTGGTAACCCCCGTGACATATCA CTTCCC788                      
TyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeuPro                          
180185190                                                                 
TATGCTCTTTTCACAGCACCAAATGTTGTGGTATGGGATGGT CAATAT836                      
TyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGlnTyr                          
195200205                                                                 
GGGTACCAAAATTTGTTTGATGCTATGTTGGATTCAGTACATGCA GCC884                      
GlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHisAlaAla                          
210215220                                                                 
ATTGATAACACTAAGATTGGTTATGTGGAGGTTGTTGTATCCGAGAGT 932                      
IleAspAsnThrLysIleGlyTyrValGluValValValSerGluSer                          
225230235240                                                              
GGGTGGCCATCAGATGGAGGATTTGCTGCCACTTATGACAACGCA CGC980                      
GlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsnAlaArg                          
245250255                                                                 
GTGTACTTAGACAATTTGGTTCGTCGTGCTAATAGAGGAAGC CCAAGA1028                     
ValTyrLeuAspAsnLeuValArgArgAlaAsnArgGlySerProArg                          
260265270                                                                 
AGGCCTTCGAAGCCCACTGAGACTTATATATTTGCCATGTTC GATGAA1076                     
ArgProSerLysProThrGluThrTyrIlePheAlaMetPheAspGlu                          
275280285                                                                 
AATCAAAAAAATCCAGAGATAGAGAAACATTTTGGGCTCTTCAAT CCC1124                     
AsnGlnLysAsnProGluIleGluLysHisPheGlyLeuPheAsnPro                          
290295300                                                                 
AACAAACAAAAAAAATACCCATTTGGGTTTGGAGGAAAGAGGCTAGGG 1172                     
AsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArgLeuGly                          
305310315320                                                              
AAAGTTGTTATTGACGACTTCAATGCAACAACTTCCATTAAGAGT GAT1220                     
LysValValIleAspAspPheAsnAlaThrThrSerIleLysSerAsp                          
325330335                                                                 
GTGTAAGGTTGAGCTCCTAAGCTTCAAAATCTCTGTTATTCCACCCATAA AAT1273                
Val                                                                       
AAGAGAGAATATGTTGTTTGTGTGAAATATGTATATATCCTTCAGTCTTGGATGAATAAA1333          
ATTTGTGAAAATTTTATTTTTTTTTTTTTTGGACTAGAAATAGCCTGATACTTAATTATT1393          
ATCTTTTTATACCACACGTT GGTTTCCTTCATGAGTACAAACCGAAATAAAACCAACAAT1453         
TAATCTTGTTTTATTACAACACACAAGCTT1483                                        
(2) INFORMATION FOR SEQ ID NO:14:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 370 amino acids                                               
(B) TYPE: amino acid                                                      
 (D) TOPOLOGY: linear                                                     
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                  
MetProSerLeuPheAlaArgAsnGlnArgPheSerLeuAlaThrLeu                          
33-30-25- 20                                                              
LeuLeuLeuLeuGluLeuLeu ThrGlyAsnLeuArgMetAlaAspAla                         
15-10-5                                                                   
GlnIleGlyValCysTyrGlyMetLeuGlyAsnAsnLeuProSerAla                          
15 1015                                                                   
AsnAspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArg                          
202530                                                                    
LeuTyrAspProAsnGlnAlaAlaLeuGluAla LeuArgAsnSerGly                         
354045                                                                    
IleGluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAla                          
505560                                                                    
ThrAsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsn                          
657075                                                                    
PheTrpProSerValLysIleLysTyrValAlaValGlyAsnGluVal                          
80 859095                                                                 
SerProValGlyGlySerSerSerValAlaGlnTyrValLeuProAla                          
100105110                                                                 
IleGlnAsnValTyr GlnAlaIleArgAlaGlnGlyLeuHisAspGln                         
115120125                                                                 
IleLysValSerThrSerIleAspMetThrLeuIleGlyAsnSerPhe                          
130 135140                                                                
ProProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAsp                          
145150155                                                                 
ProIleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeu ValAsn                         
160165170175                                                              
ValTyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeu                          
1801851 90                                                                
ProTyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGln                          
195200205                                                                 
TyrGlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHisAla                          
 210215220                                                                
AlaIleAspAsnThrLysIleGlyTyrValGluValValValSerGlu                          
225230235                                                                 
SerGlyTrpProSerAspGlyGly PheAlaAlaThrTyrAspAsnAla                         
240245250255                                                              
ArgValTyrLeuAspAsnLeuValArgArgAlaAsnArgGlySerPro                          
260 265270                                                                
ArgArgProSerLysProThrGluThrTyrIlePheAlaMetPheAsp                          
275280285                                                                 
GluAsnGlnLysAsnProGluIleGluLysHis PheGlyLeuPheAsn                         
290295300                                                                 
ProAsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArgLeu                          
305310315                                                                 
GlyLy sValValIleAspAspPheAsnAlaThrThrSerIleLysSer                         
320325330335                                                              
AspVal                                                                    
(2) INFORMATION FOR SEQ ID NO:15:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1038 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(iii) HYPOTHETICAL: NO                                                    
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 4..1017                                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                  
CATATGATTGGTGTGTGTTATGGCATGCTGGGCAACAATCTACCGTCA 48                       
MetIleGlyValCysTyrGlyMetLeuGlyAsnAsnLeuProSer                             
151015                                                                    
GCAAACGATGTTATAGGTCTTTATAGATCAAATAACATAAAGA GAATG96                       
AlaAsnAspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMet                          
202530                                                                    
AGACTCTATGATCCTAATCAAGCTGCTCTAGAAGCACTTA GAAATTCT144                      
ArgLeuTyrAspProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSer                          
354045                                                                    
GGCATTGAACTCATTCTTGGGGTGCCAAACTCTGACCTTC AAGGCCTT192                      
GlyIleGluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeu                          
505560                                                                    
GCCACCAATCCTGACACTTCTCGTCAATGGGTGCAAAAAAACG TGTTG240                      
AlaThrAsnProAspThrSerArgGlnTrpValGlnLysAsnValLeu                          
657075                                                                    
AACTTTTGGCCTAGTGTCAAAATCAAGTACGTGGCAGTTGGAAATGAA 288                      
AsnPheTrpProSerValLysIleLysTyrValAlaValGlyAsnGlu                          
80859095                                                                  
GTGAGTCCCGTTGGAGGCTCTTCTTCGGTAGCCCAATATGTTC TACCT336                      
ValSerProValGlyGlySerSerSerValAlaGlnTyrValLeuPro                          
100105110                                                                 
GCCATCCAAAATGTATACCAAGCAATAAGAGCACAAGGCC TTCATGAT384                      
AlaIleGlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAsp                          
115120125                                                                 
CAAATCAAGGTTTCAACATCTATTGACATGACCCTAATAG GAAACTCT432                      
GlnIleLysValSerThrSerIleAspMetThrLeuIleGlyAsnSer                          
130135140                                                                 
TTCCCTCCATCGCAAGGTTCCTTCAGGGGTGATGTGAGATCAT ACCTA480                      
PheProProSerGlnGlySerPheArgGlyAspValArgSerTyrLeu                          
145150155                                                                 
GATCCCATAATTGGGTACTTGGTATATGCAAATGCACCATTACTAGTC 528                      
AspProIleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeuVal                          
160165170175                                                              
AATGTGTACCCTTATTTTAGTTACACTGGTAACCCCCGTGACA TATCA576                      
AsnValTyrProTyrPheSerTyrThrGlyAsnProArgAspIleSer                          
180185190                                                                 
CTTCCCTATGCTCTTTTCACAGCACCAAATGTTGTGGTAT GGGATGGT624                      
LeuProTyrAlaLeuPheThrAlaProAsnValValValTrpAspGly                          
195200205                                                                 
CAATATGGGTACCAAAATTTGTTTGATGCTATGTTGGATT CAGTACAT672                      
GlnTyrGlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHis                          
210215220                                                                 
GCAGCCATTGATAACACTAAGATTGGTTATGTGGAGGTTGTTG TATCC720                      
AlaAlaIleAspAsnThrLysIleGlyTyrValGluValValValSer                          
225230235                                                                 
GAGAGTGGGTGGCCATCAGATGGAGGATTTGCTGCCACTTATGACAAC 768                      
GluSerGlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsn                          
240245250255                                                              
GCACGCGTGTACTTAGACAATTTGGTTCGTCGTGCTAATAGAG GAAGC816                      
AlaArgValTyrLeuAspAsnLeuValArgArgAlaAsnArgGlySer                          
260265270                                                                 
CCAAGAAGGCCTTCGAAGCCCACTGAGACTTATATATTTG CCATGTTC864                      
ProArgArgProSerLysProThrGluThrTyrIlePheAlaMetPhe                          
275280285                                                                 
GATGAAAATCAAAAAAATCCAGAGATAGAGAAACATTTTG GGCTCTTC912                      
AspGluAsnGlnLysAsnProGluIleGluLysHisPheGlyLeuPhe                          
290295300                                                                 
AATCCCAACAAACAAAAAAAATACCCATTTGGGTTTGGAGGAA AGAGG960                      
AsnProAsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArg                          
305310315                                                                 
CTAGGGAAAGTTGTTATTGACGACTTCAATGCAACAACTTCCATTAAG 1008                     
LeuGlyLysValValIleAspAspPheAsnAlaThrThrSerIleLys                          
320325330335                                                              
AGTGATGTGTAAGGTTGAGCTCCTAAGCTT 1038                                       
SerAspVal                                                                 
(2) INFORMATION FOR SEQ ID NO:16:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 338 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                  
MetIleGlyValCysTyrGlyMetLeuGlyAsnAsnLeu ProSerAla                         
151015                                                                    
AsnAspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArg                          
202530                                                                    
LeuTyrAspProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSerGly                          
354045                                                                    
IleGluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAla                          
50 5560                                                                   
ThrAsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsn                          
65707580                                                                  
PheTrpProSerValLysIl eLysTyrValAlaValGlyAsnGluVal                         
859095                                                                    
SerProValGlyGlySerSerSerValAlaGlnTyrValLeuProAla                          
100 105110                                                                
IleGlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAspGln                          
115120125                                                                 
IleLysValSerThrSerIleAspMetThrLeuIleGly AsnSerPhe                         
130135140                                                                 
ProProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAsp                          
145150155160                                                              
P roIleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeuValAsn                         
165170175                                                                 
ValTyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeu                          
 180185190                                                                
ProTyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGln                          
195200205                                                                 
TyrGlyTyrGlnAsnLeuPh eAspAlaMetLeuAspSerValHisAla                         
210215220                                                                 
AlaIleAspAsnThrLysIleGlyTyrValGluValValValSerGlu                          
225230235 240                                                             
SerGlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsnAla                          
245250255                                                                 
ArgValTyrLeuAspAsnLeuValArgArgAlaAsn ArgGlySerPro                         
260265270                                                                 
ArgArgProSerLysProThrGluThrTyrIlePheAlaMetPheAsp                          
275280285                                                                 
G luAsnGlnLysAsnProGluIleGluLysHisPheGlyLeuPheAsn                         
290295300                                                                 
ProAsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArgLeu                          
305 310315320                                                             
GlyLysValValIleAspAspPheAsnAlaThrThrSerIleLysSer                          
325330335                                                                 
AspVal                                                                    
(2) INFORMATION FOR SEQ ID NO:17:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1829 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA                                                  
(iii) HYPOTHETICAL: NO                                                    
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 438..1547                                                   
(ix) FEATURE:                                                             
(A) NAME/KEY: mat.sub.-- peptide                                          
(B) LOCATION: 537..1547                                                   
(ix) FEATURE:                                                             
(A) NAME/KEY: sig.sub.-- peptide                                          
(B) LOCATION: 438..536                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                  
AAGCTTGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCCTCAGAAGACC60            
AAAGGGCCAATTGAGACTTTTCAACAAAGGG TAATATCCGGAAACCTCCTCGGATTCCAT120          
TGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAA180           
TGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCC240           
AAAGAT GGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCT300          
TCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCAC360           
TATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGA GAGAACACGG420          
GGGACTCTAGAGGATCCATGCCTTCTCTCTTCGCTAGAAACCAGAGGTTC470                     
MetProSerLeuPheAlaArgAsnGlnArgPhe                                         
33-30 -25                                                                 
TCATTGGCTACTCTCCTGCTTCTTCTGGAACTATTGACAGGAAACCTT518                       
SerLeuAlaThrLeuLeuLeuLeuLeuGluLeuLeuThrGlyAsnLeu                          
20-15 -10                                                                 
CGCATGGCAGATGCTCAAATTGGTGTGTGTTATGGCATGCTGGGCAAC566                       
ArgMetAlaAspAlaGlnIleGlyValCysTyrGlyMetLeuGlyAsn                          
515 10                                                                    
AATCTACCGTCAGCAAACGATGTTATAGGTCTTTATAGATCAAATAAC614                       
AsnLeuProSerAlaAsnAspValIleGlyLeuTyrArgSerAsnAsn                          
15 2025                                                                   
ATAAAGAGAATGAGACTCTATGATCCTAATCAAGCTGCTCTAGAAGCA662                       
IleLysArgMetArgLeuTyrAspProAsnGlnAlaAlaLeuGluAla                          
30 3540                                                                   
CTTAGAAATTCTGGCATTGAACTCATTCTTGGGGTGCCAAACTCTGAC710                       
LeuArgAsnSerGlyIleGluLeuIleLeuGlyValProAsnSerAsp                          
4550 55                                                                   
CTTCAAGGCCTTGCCACCAATCCTGACACTTCTCGTCAATGGGTGCAA758                       
LeuGlnGlyLeuAlaThrAsnProAspThrSerArgGlnTrpValGln                          
6065 70                                                                   
AAAAACGTGTTGAACTTTTGGCCTAGTGTCAAAATCAAGTACGTGGCA806                       
LysAsnValLeuAsnPheTrpProSerValLysIleLysTyrValAla                          
758085 90                                                                 
GTTGGAAATGAAGTGAGTCCCGTTGGAGGCTCTTCTTCGGTAGCCCAA854                       
ValGlyAsnGluValSerProValGlyGlySerSerSerValAlaGln                          
95 100105                                                                 
TATGTTCTACCTGCCATCCAAAATGTATACCAAGCAATAAGAGCACAA902                       
TyrValLeuProAlaIleGlnAsnValTyrGlnAlaIleArgAlaGln                          
110 115120                                                                
GGCCTTCATGATCAAATCAAGGTTTCAACATCTATTGACATGACCCTA950                       
GlyLeuHisAspGlnIleLysValSerThrSerIleAspMetThrLeu                          
125130 135                                                                
ATAGGAAACTCTTTCCCTCCATCGCAAGGTTCCTTCAGGGGTGATGTG998                       
IleGlyAsnSerPheProProSerGlnGlySerPheArgGlyAspVal                          
140145 150                                                                
AGATCATACCTAGATCCCATAATTGGGTACTTGGTATATGCAAATGCA1046                      
ArgSerTyrLeuAspProIleIleGlyTyrLeuValTyrAlaAsnAla                          
155160165 170                                                             
CCATTACTAGTCAATGTGTACCCTTATTTTAGTTACACTGGTAACCCC1094                      
ProLeuLeuValAsnValTyrProTyrPheSerTyrThrGlyAsnPro                          
175 180185                                                                
CGTGACATATCACTTCCCTATGCTCTTTTCACAGCACCAAATGTTGTG1142                      
ArgAspIleSerLeuProTyrAlaLeuPheThrAlaProAsnValVal                          
190 195200                                                                
GTATGGGATGGTCAATATGGGTACCAAAATTTGTTTGATGCTATGTTG1190                      
ValTrpAspGlyGlnTyrGlyTyrGlnAsnLeuPheAspAlaMetLeu                          
205210 215                                                                
GATTCAGTACATGCAGCCATTGATAACACTAAGATTGGTTATGTGGAG1238                      
AspSerValHisAlaAlaIleAspAsnThrLysIleGlyTyrValGlu                          
220225 230                                                                
GTTGTTGTATCCGAGAGTGGGTGGCCATCAGATGGAGGATTTGCTGCC1286                      
ValValValSerGluSerGlyTrpProSerAspGlyGlyPheAlaAla                          
235240245 250                                                             
ACTTATGACAACGCACGCGTGTACTTAGACAATTTGGTTCGTCGTGCT1334                      
ThrTyrAspAsnAlaArgValTyrLeuAspAsnLeuValArgArgAla                          
255 260265                                                                
AATAGAGGAAGCCCAAGAAGGCCTTCGAAGCCCACTGAGACTTATATA1382                      
AsnArgGlySerProArgArgProSerLysProThrGluThrTyrIle                          
270 275280                                                                
TTTGCCATGTTCGATGAAAATCAAAAAAATCCAGAGATAGAGAAACAT1430                      
PheAlaMetPheAspGluAsnGlnLysAsnProGluIleGluLysHis                          
285290 295                                                                
TTTGGGCTCTTCAATCCCAACAAACAAAAAAAATACCCATTTGGGTTT1478                      
PheGlyLeuPheAsnProAsnLysGlnLysLysTyrProPheGlyPhe                          
300305 310                                                                
GGAGGAAAGAGGCTAGGGAAAGTTGTTATTGACGACTTCAATGCAACA1526                      
GlyGlyLysArgLeuGlyLysValValIleAspAspPheAsnAlaThr                          
315320325 330                                                             
ACTTCCATTAAGAGTGATGTGTAAGGTTGAGCTCGAATTTCCCCGATCGTT1577                   
ThrSerIleLysSerAspVal                                                     
335                                                                       
CAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCC GGTCTTGCGATGATTA1637         
TCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGT1697          
TATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAG1757          
AAAACAAAATATAGCGCGC AAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTAC1817         
TAGATCGAATTC1829                                                          
(2) INFORMATION FOR SEQ ID NO:18:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 370 amino acids                                               
(B) TYPE: amino acid                                                      
 (D) TOPOLOGY: linear                                                     
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                  
MetProSerLeuPheAlaArgAsnGlnArgPheSerLeuAlaThrLeu                          
33-30-25- 20                                                              
LeuLeuLeuLeuGluLeuLe uThrGlyAsnLeuArgMetAlaAspAla                         
15-10-5                                                                   
GlnIleGlyValCysTyrGlyMetLeuGlyAsnAsnLeuProSerAla                          
15 1015                                                                   
AsnAspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArg                          
202530                                                                    
LeuTyrAspProAsnGlnAlaAlaLeuGluAla LeuArgAsnSerGly                         
354045                                                                    
IleGluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAla                          
505560                                                                    
ThrAsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsn                          
657075                                                                    
PheTrpProSerValLysIleLysTyrValAlaValGlyAsnGluVal                          
80 859095                                                                 
SerProValGlyGlySerSerSerValAlaGlnTyrValLeuProAla                          
100105110                                                                 
IleGlnAsnValTy rGlnAlaIleArgAlaGlnGlyLeuHisAspGln                         
115120125                                                                 
IleLysValSerThrSerIleAspMetThrLeuIleGlyAsnSerPhe                          
130 135140                                                                
ProProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAsp                          
145150155                                                                 
ProIleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeu ValAsn                         
160165170175                                                              
ValTyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeu                          
180185 190                                                                
ProTyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGln                          
195200205                                                                 
TyrGlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHisAla                          
 210215220                                                                
AlaIleAspAsnThrLysIleGlyTyrValGluValValValSerGlu                          
225230235                                                                 
SerGlyTrpProSerAspGlyGl yPheAlaAlaThrTyrAspAsnAla                         
240245250255                                                              
ArgValTyrLeuAspAsnLeuValArgArgAlaAsnArgGlySerPro                          
260 265270                                                                
ArgArgProSerLysProThrGluThrTyrIlePheAlaMetPheAsp                          
275280285                                                                 
GluAsnGlnLysAsnProGluIleGluLysHis PheGlyLeuPheAsn                         
290295300                                                                 
ProAsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArgLeu                          
305310315                                                                 
GlyL ysValValIleAspAspPheAsnAlaThrThrSerIleLysSer                         
320325330335                                                              
AspVal                                                                    
(2) INFORMATION FOR SEQ ID NO:19:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 32 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                  
GATCATGAAGGCCTTGTGCTCTTATTGCTTGG32                                        
(2) INFORMATION FOR SEQ ID NO:20:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
 (A) LENGTH: 35 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                  
GAGAAGGCATGGATCCAAACATATGAATACACCAC35                                     
(2) INFORMATION FOR SEQ ID NO:21:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 38 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                  
CAGAGATTTTGAAGCTTAGGAGCTCAACCTTACACATC 38                                 
(2) INFORMATION FOR SEQ ID NO:22:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 44 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                  
TATGATTGGTGTGTGTTATGGCATGACTAACCAC ACACAATACC44                           
(2) INFORMATION FOR SEQ ID NO:23:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 9 amino acids                                                 
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                  
MetIleGlyValXaaTyrGlyMetLeu                                               
 15                                                                       
(2) INFORMATION FOR SEQ ID NO:24:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 14 amino acids                                                
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: peptide                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                  
MetIleGlyValXaaTyrGlyMetLeuGlyAsnAsnLeuPro                                
1510                                                                      
(2) INFORMATION FOR SEQ ID NO:25:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 337 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                  
IleGlyValCysTyrGlyMetLeuGl yAsnAsnLeuProSerAlaAsn                         
151015                                                                    
AspValIleGlyLeuTyrArgSerAsnAsnIleLysArgMetArgLeu                          
2025 30                                                                   
TyrAspProAsnGlnAlaAlaLeuGluAlaLeuArgAsnSerGlyIle                          
354045                                                                    
GluLeuIleLeuGlyValProAsnSerAspLeuGlnGlyLeuAlaTh r                         
505560                                                                    
AsnProAspThrSerArgGlnTrpValGlnLysAsnValLeuAsnPhe                          
65707580                                                                  
TrpProSerVa lLysIleLysTyrValAlaValGlyAsnGluValSer                         
859095                                                                    
ProValGlyGlySerSerSerValAlaGlnTyrValLeuProAlaIle                          
100 105110                                                                
GlnAsnValTyrGlnAlaIleArgAlaGlnGlyLeuHisAspGlnIle                          
115120125                                                                 
LysValSerThrSerIleAspMetThrLeu IleGlyAsnSerPhePro                         
130135140                                                                 
ProSerGlnGlySerPheArgGlyAspValArgSerTyrLeuAspPro                          
145150155 160                                                             
IleIleGlyTyrLeuValTyrAlaAsnAlaProLeuLeuValAsnVal                          
165170175                                                                 
TyrProTyrPheSerTyrThrGlyAsnProArgAspIleSerLeuP ro                         
180185190                                                                 
TyrAlaLeuPheThrAlaProAsnValValValTrpAspGlyGlnTyr                          
195200205                                                                 
GlyTyrGlnAsn LeuPheAspAlaMetLeuAspSerValHisAlaAla                         
210215220                                                                 
IleAspAsnThrLysIleGlyTyrValGluValValValSerGluSer                          
225230 235240                                                             
GlyTrpProSerAspGlyGlyPheAlaAlaThrTyrAspAsnAlaArg                          
245250255                                                                 
ValTyrLeuAspAsnLeuValArgArg AlaAsnArgGlySerProArg                         
260265270                                                                 
ArgProSerLysProThrGluThrTyrIlePheAlaMetPheAspGlu                          
275280 285                                                                
AsnGlnLysAsnProGluIleGluLysHisPheGlyLeuPheAsnPro                          
290295300                                                                 
AsnLysGlnLysLysTyrProPheGlyPheGlyGlyLysArgLeuGly                          
305 310315320                                                             
LysValValIleAspAspPheAsnAlaThrThrSerIleLysSerAsp                          
325330335                                                                 
Val                                                                       
(2) INFORMATION FOR SEQ ID NO:26:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 370 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                  
MetAspThrSerHisLysHisIleAlaLeuGlnMetAlaAlaIleIle                          
15 1015                                                                   
LeuLeuGlyLeuLeuValSerSerThrGluIleValGlyAlaGlnSer                          
202530                                                                    
ValGlyValCysTyrGlyMetLeuGly AsnAsnLeuProProAlaSer                         
354045                                                                    
GlnValValGlnLeuTyrLysSerLysAsnIleArgArgMetArgLeu                          
505560                                                                    
 TyrAspProAsnGlnAlaAlaLeuGlnAlaLeuArgGlySerAsnIle                         
65707580                                                                  
GluValMetLeuGlyValProAsnSerAspLeuGlnAsnIleAlaAla                          
 859095                                                                   
AsnProSerAsnAlaAsnAsnTrpValGlnArgAsnValArgAsnPhe                          
100105110                                                                 
TrpProAlaVal LysPheArgTyrIleAlaValGlyAsnGluValSer                         
115120125                                                                 
ProValThrGlyThrSerSerLeuThrArgTyrLeuLeuProAlaMet                          
130135 140                                                                
ArgAsnIleArgAsnAlaIleSerSerAlaGlyLeuGlnAsnAsnIle                          
145150155160                                                              
LysValSerSerSerValAspMetThrLeuIle GlyAsnSerPhePro                         
165170175                                                                 
ProSerGlnGlySerPheArgAsnAspValArgSerPheIleAspPro                          
180185 190                                                                
IleIleGlyPheValArgArgIleAsnSerProLeuLeuValAsnIle                          
195200205                                                                 
TyrProTyrPheSerTyrAlaGlyAsnProArgAspIleSerLeuPro                          
 210215220                                                                
TyrAlaLeuPheThrAlaProAsnValValValGlnAspGlySerLeu                          
225230235240                                                              
GlyTyrArgAsnLeu PheAspAlaMetSerAspAlaValTyrAlaAla                         
245250255                                                                 
LeuSerArgAlaGlyGlyGlySerIleGluIleValValSerGluSer                          
260 265270                                                                
GlyTrpProSerAlaGlyAlaPheAlaAlaThrThrAsnAsnAlaAla                          
275280285                                                                 
ThrTyrTyrLysAsnLeuIleGlnHisValLys ArgGlySerProArg                         
290295300                                                                 
ArgProAsnLysValIleGluThrTyrLeuPheAlaMetPheAspGlu                          
3053103153 20                                                             
AsnAsnLysAsnProGluLeuGluLysHisPheGlyLeuPheSerPro                          
325330335                                                                 
AsnLysGlnProLysTyrProLeuSerPheGlyPheSerAspArgTyr                          
 340345350                                                                
TrpAspIleSerAlaGluAsnAsnAlaThrAlaAlaSerLeuIleSer                          
355360365                                                                 
GluMet                                                                    
370

Claims (25)

We claim
1. A DNA construct, which codes for a protein having β-1,3-glucanase activity, wherein said protein comprises the following sequence (a1) (SEQ ID NO. 1) ##STR1##
2. A DNA construct according to claim 1, further comprising immediately downstream of the nucleotide sequence coding for the amino acid sequence (a1) (SEQ ID NO. 1), the nucleotide sequence coding for the amino acid sequence (a4) (SEQ ID NO. 2) below:
______________________________________                                    
Tyr Pro Phe Gly Phe Gly Gly Lys Arg Leu Gly Lys                           
Val Val Ile Asp Asp Phe Asn Ala Thr Thr Ser Ile Lys Ser                   
Asp Val                                                                   
______________________________________
truncated in its carboxy-terminal portion by 0 to 27 amino acids.
3. A DNA construct according to claim 2, further comprising immediately upstream of the nucleotide sequence coding for the amino acid sequence (a1) (SEQ ID NO. 1), a codon for Gln.
4. A DNA construct according to claim 3, which codes for a protein having β-1,3-glucanase activity, which comprises the following sequence (a5) (SEQ ID NO. 3) ##STR2##
5. A DNA construct according to claim 3 which contains, upstream of said codon for Gln preceding the nucleotide sequence coding for the amino acid sequence (a1) (SEQ ID NO. 1), a region encoding a signal sequence.
6. A DNA construct according to claim 5, wherein said region encoding a signal sequence encodes a signal peptide of the following sequence (a2) (SEQ ID NO. 7)
______________________________________                                    
Met Pro Ser Leu Phe Ala Arg Asn Gln Arg Phe Ser Leu Ala                   
Thr Leu Leu Leu Leu Leu Glu Leu Leu Thr Gly Asn Leu Arg                   
Met Ala Asp Ala.                                                          
______________________________________
7. A DNA construct according to claim 1, wherein the nucleotide sequence coding for the amino acid sequence (a1) (SEQ ID NO. 1) is the following sequence (Na1) (SEQ ID NO. 8)
__________________________________________________________________________
ATTGGTGTGT GTTATGGCAT GCTGGGCAAC AATCTACCGT CAGCAAACGA                    
TGTTATAGGT CTTTATAGAT CAAATAACAT AAAGAGAATG AGACTCTATG                    
ATCCTAATCA AGCTGCTCTA GAAGCACTTA GAAATTCTGG CATTGAACTC                    
ATTCTTGGGG TGCCAAACTC TGACCTTCAA GGCCTTGCCA CCAATCCTGA                    
CACTTCTCGT CAATGGGTGC AAAAAAACGT GTTGAACTTT TGGCCTAGTG                    
TCAAAATCAA GTACGTGGCA GTTGGAAATG AAGTGAGTCC CGTTGGAGGC                    
TCTTCTTCGG TAGCCCAATA TGTTCTACCT GCCATCCAAA ATGTATACCA                    
AGCAATAAGA GCTCAAGGCC TTCATGATCA AATCAAGGTT TCAACATCTA                    
TTGACATGAC CCTAATAGGA AACTCTTTCC CTCCATCGCA AGGTTCCTTC                    
AGGGGTGATG TGAGATCATA CCTAGATCCC ATAATTGGGT ACTTGGTATA                    
TGCAAATGCA CCATTACTAG TCAATGTGTA CCCTTATTTT AGTTACACTG                    
GTAACCCCCG TGACATATCA CTTCCCTATG CTCTTTTCAC AGCACCAAAT                    
GTTGTGGTAT GGGATGGTCA ATATGGGTAC CAAAATTTGT TTGATGCTAT                    
GTTGGATTCA GTACATGCAG CCATTGATAA CACTAAGATT GGTTATGTGG                    
AGGTTGTTGT ATCCGAGAGT GGGTGGCCAT CAGATGGAGG ATTTGCTGCC                    
ACTTATGACA ACGCACGCGT GTACTTAGAC AATTTGGTTC GTCGTGCTAA                    
TAGAGGAAGC CCAAGAAGGC CTTCGAAGCC CACTGAGACT TATATATTTG                    
CCATGTTCGA TGAAAATCAA AAAAATCCAG AGATAGAGAA ACATTTTGGG                    
CTCTTCAATC CCAACAAACA AAAAAAA.                                            
__________________________________________________________________________
8. A DNA construct according to claim 6, wherein the nucleotide sequence coding for the amino acid sequence (a2) (SEQ ID NO. 7) is the following sequence (Na2) (SEQ ID NO. 9)
__________________________________________________________________________
ATGCCTTCTC TCTTCGCTAG AAACCAGAGG TTCTCATTGG CTACTCTCCT                    
GCTTCTTCTG GAACTATTGA CAGGAAACCT TCGCATGGCA GATGCT.                       
__________________________________________________________________________
9. A DNA construct according to claim 2 wherein the nucleotide sequence coding for the amino acid sequence (a4) (SEQ ID NO. 2) is the following sequence (Na4) (SEQ ID NO. 10)
__________________________________________________________________________
TACCCATTTG GGTTTGGAGG AAAGAGGCTA GGGAAAGTTG TTATTGACGA                    
CTTCAATGCA ACAACTTCCA TTAAGAGTGA TGTG.                                    
__________________________________________________________________________
10. A bacterium which contains a DNA construct according to claim 1 operably linked to cis-acting signals effective for its replication and its expression in said bacterium.
11. A plant cell transformed with a DNA construct according to claim 1 operably linked to cis-acting signals effective for its expression in said cell.
12. A plant cell according to claim 11, which belongs to a species selected from the group consisting of Nicotiana tabacum, Helianthus annuus and Brassica napus.
13. A plant or plant part, which plant or plant part contains a DNA construct according to claim 1 operably linked to cis-acting signals effective for its expression in said plant or plant part.
14. A plant or plant part according to claim 13, which plant or plant part belongs to a species selected from the group consisting of Nicotiana tabacum, Helianthus annuus and Brassica napus.
15. A plant part according to claim 13, which is capable of forming a complete new plant or of producing seeds.
16. A plant part according to claim 15, which is a seed.
17. An isolated, purified protein having β-1,3-glucanase activity, which comprises a region having the amino acid sequence (a1) (SEQ ID NO. 1)
______________________________________                                    
Ile Gly Val Cys Tyr Gly Met Leu Gly Asn Asn Leu Pro Ser                   
Ala Asn Asp Val Ile Gly Leu Tyr Arg Ser Asn Asn Ile Lys                   
Arg Met Arg Leu Tyr Asp Pro Asn Gln Ala Ala Leu Glu Ala                   
Leu Arg Asn Ser Gly Ile Glu Leu Ile Leu Gly Val Pro Asn                   
Ser Asp Leu Gln Gly Leu Ala Thr Asn Pro Asp Thr Ser Arg                   
Gln Trp Val Gln Lys Asn Val Leu Asn Phe Trp Pro Ser Val                   
Lys Ile Lys Tyr Val Ala Val Gly Asn Glu Val Ser Pro Val                   
Gly Gly Ser Ser Ser Val Ala Gln Tyr Val Leu Pro Ala Ile                   
Gln Asn Val Tyr Gln Ala Ile Arg Ala Gln Gly Leu His Asp                   
Gln Ile Val Ser Thr Ser Ile Asp Met Thr Leu Ile Gly                       
Asn Ser Phe Pro Pro Ser Gln Gly Ser Phe Arg Gly Asp Val                   
Arg Ser Tyr Leu Asp Pro Ile Ile Gly Tyr Leu Val Tyr Ala                   
Asn Ala Pro Leu Leu Val Asn Val Tyr Pro Tyr Phe Ser Tyr                   
Thr Gly Asn Pro Arg Asp Ile Ser Leu Pro Tyr Ala Leu Phe                   
Thr Ala Pro Asn Val Val Val Trp Asp Gly Gln Tyr Gly Tyr                   
Gln Asn Leu Phe Asp Ala Met Leu Asp Ser Val His Ala Ala                   
Ile Asp Asn Thr Lys Ile Gly Tyr Val Glu Val Val Val Ser                   
Glu Ser Gly Trp Pro Ser Asp Gly Gly Phe Ala Ala Thr Tyr                   
Asp Asn Ala Arg Val Tyr Asp Asn Leu Val Arg Arg Ala                       
Asn Arg Gly Ser Pro Arg Arg Pro Ser Lys Pro Thr Glu Thr                   
Tyr Ile Phe Ala Met Phe Asp Glu Asn Gln Lys Asn Pro Glu                   
Ile Glu Lys His Phe Gly Leu Phe Asn Pro Asn Lys Gln Lys                   
Lys.                                                                      
______________________________________
18. An isolated, purified protein according to claim 17, further comprising immediately downstream of the sequence (a1) (SEQ ID NO. 1), a region having the amino acid sequence (a4) (SEQ ID NO. 2)
______________________________________                                    
Tyr Pro Phe Gly Phe Gly Gly Lys Arg Leu Gly Lys                           
Val Val Ile Asp Asp Phe Asn Ala Thr Thr Ser Ile Lys Ser                   
Asp Val.                                                                  
______________________________________
truncated in its carboxy-terminal portion by 0 to 27 amino acids.
19. An isolated, purified protein having β-1,3-glucanase activity, comprising a region having the amino acid sequence (a5) (SEQ ID NO. 3)
______________________________________                                    
Gln Ile Gly Val Cys Tyr Gly Met Leu Gly Asn Asn Leu Pro                   
Ser Ala Asn Asp Val Ile Gly Leu Tyr Arg Ser Asn Asn Ile                   
Lys Arg Met Arg Leu Tyr Asp Pro Asn Gln Ala Ala Leu Glu                   
Ala Leu Arg Asn Ser Gly Ile Glu Leu Ile Leu Gly Val Pro                   
Asn Ser Asp Leu Gln Gly Leu Ala Thr Asn Pro Asp Thr Ser                   
Arg Gln Trp Val Gln Lys Asn Val Leu Asn Phe Trp Pro Ser                   
Val Lys Ile Lys Tyr Val Ala Val Gly Asn Glu Val Ser Pro                   
Val Gly Gly Ser Ser Ser Val Ala Gln Tyr Val Leu Pro Ala                   
Ile Gln Asn Val Tyr Gln Ala Ile Arg Ala Gly Gly Leu His                   
Asp Gln Ile Lys Val Ser Thr Ser Ile Asp Met Thr Leu Ile                   
Gly Asn Ser Phe Pro Pro Ser Gln Gly Ser Phe Arg Gly Asp                   
Val Arg Ser Tyr Leu Asp Pro Ile Ile Gly Tyr Leu Val Tyr                   
Ala Asn Ala Pro Leu Leu Val Asn Val Tyr Pro Tyr Phe Ser                   
Tyr Thr Gly Asn Pro Arg Asp Ile Ser Leu Pro Tyr Ala Leu                   
Phe Thr Ala Pro Asn Val Val Val Trp Asp Gly Gln Tyr Gly                   
Tyr Gln Asn Leu Phe Asp Ala Met Leu Asp Ser Val His Ala                   
Ala Ile Asp Asn Thr Lys Ile Gly Tyr Val Glu Val Val Val                   
Ser Glu Ser Gly Trp Pro Ser Asp Gly Gly Phe Ala Ala Thr                   
Tyr Asp Asn Ala Arg Val Tyr Leu Asp Asn Leu Val Arg Arg                   
Ala Asn Arg Gly Ser Pro Arg Arg Pro Ser Lys Pro Thr Glu                   
Thr Tyr Ile Phe Ala Met Phe Asp Glu Asn Gln Lys Asn Pro                   
Glu Ile Glu Lys His Phe Gly Leu Phe Asn Pro Asn Lys Gln                   
Lys Lys Tyr Pro Phe Gly Phe Gly Gly Lys Arg Leu Gly Lys                   
Val Val Ile Asp Asp Phe Asn Ala Thr Thr Ser Ile Lys Ser                   
Asp Val.                                                                  
______________________________________
20. An isolated, purified protein according to claim 17, which possesses an apparent molecular mass of 36±3 kDa.
21. An isolated, purified protein according to claim 17, which possesses an apparent molecular mass of 37±3 kDa.
22. An isolated, purified protein according to claim 17, which possesses an apparent molecular mass of 39±3 kDa.
23. A method for preparing a protein according to claim 17, which comprises culturing a bacterium of claim 10.
24. A method for preparing a protein according to claim 17, which comprises culturing a plant cell of claim 11.
25. A method for preparing a protein according to claim 17, which comprises culturing a plant or plant part of claim 13.
US07/966,187 1991-03-25 1992-03-25 Recombinant DNA coding for a novel protein having β-1,3-glucanase activity, bacteria containing this DNA, transformed plant cells and plants Expired - Lifetime US5477001A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR919103588A FR2674538B1 (en) 1991-03-25 1991-03-25 RECOMBINANT DNA ENCODING A NOVEL PROTEIN WITH BETA 1,3-GLUCANASE BACTERIA ACTIVITY CONTAINING THIS DNA, PLANT CELLS AND TRANSFORMED PLANTS.
FR9103588 1991-03-25
PCT/FR1992/000268 WO1992016632A1 (en) 1991-03-25 1992-03-25 Recombinant dna coding for a novel protein having beta-1,3-glucanase activity of soya

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US20060037505A1 (en) * 2002-08-07 2006-02-23 Avigdor Bieber Lithographic printing memebers and a method and a system for preparation of lithographic printing members
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US5952548A (en) * 1997-11-04 1999-09-14 Iowa State University Research Foundation, Inc. Soybean glucanases, compounds which encode therefor and related methods
WO2004005500A1 (en) * 2002-07-02 2004-01-15 Genomine Inc. Novel polypeptide having function of 7-keto-8-aminopelargonic acid synthase of plant and method for inducing growth inhibition and lethality by suppressing expression of the polypeptide
KR100510430B1 (en) * 2002-07-02 2005-08-26 제노마인(주) Novel polypeptide having function of 7-keto-8-aminopelargonic acid synthase of plant and method for inducing growth inhibition and lethality by suppressing expression of the polypeptide
US20060037505A1 (en) * 2002-08-07 2006-02-23 Avigdor Bieber Lithographic printing memebers and a method and a system for preparation of lithographic printing members
US20150268276A1 (en) * 2014-03-24 2015-09-24 Hitachi Metals, Ltd. Probe cable and harness using the same

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